System for imaging and orienting seeds and method of use

ABSTRACT

A system and method for the automated or semi-automated imagining and orienting of seeds to prepare the seeds for transformation and transgenic engineering.

This application is a continuation of U.S. patent application Ser. No.14/704,691, now U.S. Pat. No. 9,924,626, filed on May 5, 2015, whichclaims priority to U.S. Provisional Patent Application Ser. No.61/989,266, filed on May 6, 2014, the entire disclosures of both ofwhich are expressly incorporated herein by reference.

CROSS-REFERENCE TO RELATED U.S. PATENT APPLICATION

Cross-reference is made to U.S. Provisional Patent Application Ser. No.61/989,275 entitled “SYSTEM FOR SEED PREPARATION AND METHOD OF USE” byDonald L. McCarty, II et al., which was filed on May 6, 2014; and toU.S. Provisional Patent Application Ser. No. 61/989,276 entitled “SYSTEMFOR CUTTING AND PREPARING SEEDS AND METHOD OF USE” by Donald L. McCarty,II et al., which was filed on May 6, 2014, each of which is expresslyincorporated herein by reference.

TECHNICAL FIELD

The present disclosure relates generally to devices for preparing seedsfor use in plant breeding, and, more specifically, to a device forpreparing seeds and seed explants for gene transformation and transgenicengineering.

BACKGROUND

Soybean (Glycine max) is one of the most important agricultural crops,with an annual crop yield of more than 200 million metric tons, and anestimated value exceeding 40 billion U.S. dollars worldwide. Soybeanaccounts for over 97% of all oilseed production globally. Thus, reliableand efficient methods for improving the quality and yield of thisvaluable crop are of significant interest.

Traditional breeding methods for improving soybean have been constrainedbecause the majority of soybean cultivars are derived from only a fewparental lines, leading to a narrow germplasm base for breeding.Christou et al., TIBTECH 8:145-151 (1990). Modern research efforts havefocused on plant genetic engineering techniques to improve soybeanproduction. Transgenic methods are designed to introduce desired genesinto the heritable germline of crop plants to generate elite plantlines. The approach has successfully increased the resistance of severalother crop plants to disease, insects, and herbicides, while improvingnutritional value.

Several methods have been developed for transferring genes into planttissue, including high velocity microprojection, microinjection,electroporation, and direct DNA uptake. Agrobacterium-mediated genetransformation has more recently been used to introduce genes ofinterest into soybeans. However, soybeans have proven to be achallenging system for transgenic engineering. Efficient transformationand regeneration of soybean explants is difficult to achieve, andfrequently hard to repeat.

Agrobacterium tumefaciens, a pathogenic, soil-dwelling bacterium, hasthe inherent ability to transfer its DNA, called T-DNA, into host plantcells and to induce the host cells to produce metabolites useful forbacterial nutrition. Using recombinant techniques, some or all of theT-DNA may be replaced with a gene or genes of interest, creating abacterial vector useful for transforming the host plant.Agrobacterium-mediated gene transfer is typically directed atundifferentiated cells in tissue culture, but may also be directed atdifferentiated cells taken from the leaf or stem of the plant. A numberof procedures have been developed for Agrobacterium-mediatedtransformation of soybean, which may loosely be classified based on theexplant tissue subjected to transformation.

U.S. Pat. No. 7,696,408, Olhoft, et al., discloses a cotyledonary nodemethod for transforming both monocotyledonous and dicotyledonous plants.The “cot node” method involves removing the hypocotyl from 5-7 day oldsoybean seedlings by cutting just below the cotyledonary node, splittingand separating the remaining hypocotyl segment with the cotyledons, andremoving the epicotyl from the cotyledon. The cotyledonary explant iswounded in the region of the axillary bud and/or cotyledonary node, andcultivated with Agrobacterium tumefaciens for five days in the dark. Themethod requires in-vitro germination of the seeds, and the wounding stepintroduces significant variability.

U.S. Pat. No. 6,384,301, Martinelli et al., disclosesAgrobacterium-mediated gene delivery into living meristem tissue fromsoybean embryos excised from soybean seeds, followed by culturing of themeristem explant with a selection agent and hormone to induce shootformation. Like the “cot node” method, the meristem explants arepreferably wounded prior to infection.

U.S. Pat. No. 7,473,822, Paz et al., discloses a modified cotyledonarynode method called the “half-seed explant” method. Mature soybean seedsare imbibed, surface-sterilized and split along the hilum. Prior toinfection, the embryonic axis and shoots are completely removed, but noother wounding occurs. Agrobacterium-mediated transformation proceeds,potential transformants are selected, and explants are regenerated onselection medium.

Transformation efficiencies remain relatively low with these methods, onthe order of 0.3% to 2.8% for the “cot node” method, 1.2 to 4.7% for the“meristem explant” method, and between 3.2% and 8.7% (overall 4.9%) forthe “half-seed explant” method. Transformation efficiencies ofapproximately 3% are typical in the art.

An improved “split-seed” transgenic protocol may accelerate futureproduction and development of transgenic soybean products. An efficientand high-throughput method for stable integration of a transgene intosoybean tissue would facilitate breeding programs and have the potentialto increase crop productivity.

SUMMARY

A method and apparatus for automated seed preparation is disclosed.According to one aspect, the method includes locating the seed on asurface or container, engaging the seed with an automated tool,orienting the seed for cutting or wounding, and cutting or wounding theseed when the seed is oriented. The method may also include partiallycutting the embryonic axis of the seed. In some embodiments, the cut orwounded seed is transformed with exogenous DNA.

The scope of the disclosure is not limited to the specified structuresor the specific terms used. For example, the term “robotic arm” may besubstituted with the term “automated tool.” Additionally, the terms“surface” or “container” may be substituted for the term “tray,” and theterm “cutting block” may be substituted with the terms “cuttingsurface,” “support block,” or “block.”

The automated seed preparation method may include capturing an image ofa tray including at least one seed, locating the seed on the surface orcontainer (e.g., the tray) based on a captured image, gripping the seedwith an automated tool (e.g., the robotic arm), orienting the seed on acutting surface (e.g., the cutting block) for bisection of the seed, andbisecting the seed when the seed is oriented on the cutting surface. Insome embodiments, the method may also include partially cutting theembryonic axis of the seed. In some embodiments, locating the seed maycomprise locating the seed on a tray having a plurality of seeds placedthereon.

In some embodiments, the method may further comprise operating therobotic arm to move the seed from the tray to a separate location,capturing a plurality of images of the seed in the separate location,and determining a proper orientation of the seed for bisection based onthe plurality of captured images. In some embodiments, the plurality ofimages may be captured by one camera that captures an image set from oneor more perspectives. In other embodiments, capturing the plurality ofimages may comprise operating a first camera to capture a first imageset of the seed from a first perspective, and operating a second camerato capture a second image set of the seed from a second perspectivedifferent from the first perspective. As used herein, an image set mayinclude one image or a plurality of images.

Additionally, in some embodiments, determining the proper orientation ofthe seed may comprise locating a center of a hilum of the seed and alongitudinal axis of the seed.

In some embodiments, orienting the seed on the cutting surface (e.g.,the cutting block) for bisection of the seed may comprise aligning theseed with a cutting blade of a cutting device along an imaginary planedefined by the center of the hilum of the seed and the longitudinal axisof the seed.

In some embodiments, the method may further comprise trimming anembryonic axis of the seed when the seed is located on the cuttingsurface. In some embodiments, determining the proper orientation of theseed for bisection based on the plurality of captured images may alsoinclude determining the proper orientation of the seed for trimming theembryonic axis. Trimming the embryonic axis of the seed may includepositioning a cutting blade perpendicular to a longitudinal axis of theseed.

In some embodiments, bisecting the seed on the cutting surface maycomprise bisecting the seed after trimming the embryonic axis of theseed. Additionally, in some embodiments, bisecting the seed on thecutting surface may comprise cutting through less than an entirety ofthe seed. In some embodiments, the method may further comprise movingthe bisected seed to an Agrobacterium tumefaciens solution.

In some embodiments, the method may comprise sterilizing a grip of theautomated tool (e.g., the robotic arm) prior to gripping the seed. Themethod may comprise operating the automated tool or robotic arm toselect a cutting blade, and positioning the cutting blade on a cuttingdevice. The method may further comprise bisecting the seed when the seedis oriented on the cutting block by inserting the cutting blade into theseed. In some embodiments, the method may include gripping the cuttingblade with the same or a different automated tool after bisecting theseed and operating the automated tool to replace the cutting blade witha second cutting blade on the cutting device.

According to another aspect, a seed preparation apparatus comprises afirst camera configured to capture a first image set of a seed placed ona surface or a tray, a robotic arm operable to grip the seed and to movethe seed from the surface or the tray to a lighted chamber, a secondcamera configured to capture a second image set of the seed within thelighted chamber, and a cutting block configured to receive the seed. Therobotic arm is further operable to position the seed on the cuttingblock in a proper orientation for bisection of the seed.

In some embodiments, the seed preparation apparatus may comprise a lightsource positioned on a first side of the tray to illuminate the seeds onthe tray. In some embodiments, the lighted chamber may be defined in alighted dome.

In some embodiments, the seed preparation apparatus may comprise a thirdcamera configured to capture a third image set of the seed within thelighted chamber, and an electronic controller configured to analyze thesecond image set and the third image set to determine the properorientation of the seed.

In some embodiments, the seed preparation apparatus may comprise a lightsource configured to light an interior of the lighted chamber.

In some embodiments, the electronic controller may be further configuredto analyze the first image set to locate the seed on the tray.

According to another aspect, a seed preparation apparatus comprises achamber, a first camera configured to capture a first image set of aseed on a tray, a second camera configured to capture a second image setof the seed within the chamber, a cutting device configured to bisectthe seed, a robotic arm including a gripping device to grip the seed formovement, and an electronic controller. The electronic controller isconfigured to locate the seed on the tray based on the first image set,operate the robotic arm to grasp the seed on the tray and move the seedto the cutting device in an orientation based on the second image set,and operate the cutting device to bisect the seed.

In some embodiments, the electronic controller may be configured tooperate the robotic arm to move the seed from the tray to the chamber,and operate the second camera to capture the second image set.

In some embodiments, the electronic controller may be configured toanalyze a plurality of images of the seed to determine a properorientation of the seed for bisection of the seed and trimming anembryonic axis of the seed.

In some embodiments, the robotic arm may be configured to move the seedto the cutting device to position the seed in the proper orientation,and the cutting device is configured to trim the embryonic axis of theseed while the seed is positioned in the cutting device in the properorientation.

According to another aspect of the disclosure, a cutting block isdisclosed. The cutting block comprises a body including a front wall anda substantially planar upper wall extending away from the front wall. Afirst opening is defined in the front wall, a second opening is definedin the upper wall, and a plurality of inner walls extend inwardly fromthe first opening and the second opening to define a slot in the frontwall and the upper wall. The slot is sized to receive a cutting tool.The cutting block is sized to support a seed such as a soybean seed orany seed of that size that cutting tool may be advanced along the slotinto contact with the seed.

In some embodiments, the upper wall may extend from the front wall to arear edge. The body may further include a substantially planar side wallextending upwardly from the rear edge. In some embodiments, the sidewall may be a first side wall extending from the rear edge of the upperwall to an upper edge The body may further include a second side wallextending from the upper edge of the first side wall. The second sidewall may extend obliquely relative to the first side wall and the upperwall of the body.

In some embodiments, the second side wall may extend from the upper edgeof the first side wall to a top edge, and the body may further include atop wall extending from the top edge of the second side wall. The topwall may extend obliquely relative to the second side wall.

In some embodiments, the top wall may extend parallel to the upper wallof the cutting block.

In some embodiments, the slot may extend from the first opening in thefront wall to a back edge positioned between the front wall and the rearedge of the upper wall.

In some embodiments, the first opening may be positioned in a center ofthe front wall. In some embodiments, the body may be formed as a singlemonolithic metallic body. In some embodiments, the body may be securedto a surface with an automated cutting system.

In further embodiments, a combination is disclosed. The combinationincludes each cutting block herein with a seed such as a soybean seed oany seed of that size. The soybean seed may be cut, bisected, trimmed,or otherwise wounded for transformation. In some embodiments, theembryonic axis of the seed may be trimmed for transformation.

According to another aspect, a cutting system is disclosed. The cuttingsystem includes an automated cutting system including a cutting tool,and a cutting block including an upper wall and a slot defined in theupper wall that is sized to receive the cutting tool of the automatedcutting system. The automated cutting system is operable to move thecutting tool linearly along a first axis relative to the cutting block,and rotate the cutting tool about the first axis to position the cuttingtool for insertion into the slot.

In some embodiments, the automated cutting system may further include anelectric motor operable to move the cutting tool linearly along thefirst axis, and a pneumatic device operable to rotate the cutting toolabout the first axis.

In some embodiments, the automated cutting system further may include apair of movable jaws configured to receive the cutting tool. The pair ofmovable jaws may be operable to move between an unlocked position inwhich the cutting tool is removable from the jaws, and a locked positionin which the cutting tool is retained on the jaws.

In some embodiments, the automated cutting system further may include asecond pneumatic device operable to move the pair of jaws between theunlocked position and the locked position.

In some embodiments, the automated cutting system further may include anelectronic controller including a processor, a memory device, and aplurality of instructions stored in the memory device, which, whenexecuted by the processor, cause the processor to operate a firstcompressed air source to move the pair of jaws from the unlockedposition to the locked position, operate a second compressed air sourceto rotate the cutting tool about the first axis to an orientation inwhich the cutting tool extends vertically, and operate the firstelectric motor to advance the cutting tool into the slot defined in thecutting block. In some embodiments, the electronic controller mayfurther include a plurality of instruction, which, when executed by theprocessor, cause the processor to operate the first electric motor toremove the cutting tool from the slot defined in the cutting block,operate the second compressed air source to rotate the cutting toolabout the first axis to a second orientation in which the cutting toolextends horizontally, and operate the first electric motor to advancethe cutting tool over the upper wall of the cutting block.

In some embodiments, the cutting block may include a front wall and thesubstantially planar upper wall extends away from the front wall, and afirst opening is defined in the front wall, a second opening is definedin the upper wall, and a plurality of inner walls extend inwardly fromthe first opening and the second opening to define the slot in the frontwall and the upper wall.

In some embodiments, the cutting tool may be removably coupled to theautomated cutting system.

According to another aspect, a method of cutting a seed is disclosed.The method includes advancing a cutting tool along a first axis into aslot defined in a cutting block and to make a first cut in the seed,rotating the cutting tool about the first axis, and advancing thecutting tool into the seed to make a second cut. In some embodiments,the cutting tool may be rotated by operating a compressed air source oran electric motor. In some embodiments, the cutting tool may be advancedinto the seed by operating one or more electric motors.

In some embodiments, the method may comprise positioning the cuttingtool on a pair of jaws, and moving the pair of jaws to secure thecutting tool to the pair of jaws. In some embodiments, the pair of jawsmay be moved apart to engage the cutting tool. Additionally, in someembodiments, the pair of jaws may be moved by operating a compressed airsource.

In some embodiments, positioning the cutting tool on the pair of jawsmay include attaching the cutting tool to an automated tool such as arobotic arm.

In some embodiments, the method may further comprise operating anegative pressure source to attach the cutting tool to the robotic armvia suction.

According to another aspect, a method for imaging a seed is disclosed.The method includes using an automated tool such as a robotic arm toposition a seed including a hilum within a lighted structure such as adome, projecting the seed onto a first plane extending perpendicular toa center axis of the lighted dome, rotating the seed to orient the seedparallel to a first imaginary horizontal line positioned in the firstplane, projecting the seed onto a second plane extending perpendicularto the first plane, orienting the seed parallel to a second imaginaryhorizontal line positioned in the second plane, identifying a distancebetween the hilum of the seed and the second imaginary horizontal line,and orienting the seed to position the hilum on the second imaginaryhorizontal line based on the identified distance.

As described above, the scope of the disclosure is not limited to thedisclosed structures or terms used. Thus, the term “lighted dome” may besubstituted with, for example, the term “lighted structure.”

In some embodiments, orienting the seed to position the hilum on thesecond imaging horizontal line may comprise orienting the seed toposition a center of the hilum on the second imaginary horizontal line.In some embodiments, orienting the seed to position the center of thehilum on the second imaginary horizontal line may comprise orienting theseed such that a center of mass of the hilum is coincident with a centerof mass of the seed.

Additionally, in some embodiments, the method may further compriseidentifying a location of the embryo of the seed, identifying an edge ofthe hilum nearest the identified location and an outer edge of the seedalong the second imaginary horizontal line, and identifying a pointbetween the edge of the hilum and the outer edge of the seed at which totrim an embryonic axis of the seed. In some embodiments, identifying thelocation of the embryo may comprise analyzing one or more projections ofthe seed onto the second plane using feature matching.

In some embodiments, projecting the seed onto the first plane maycomprise capturing a first image set with a first camera, and projectingthe seed on to a second plane may comprise capturing a second image setwith a second camera.

In some embodiments, the first camera may have an optical axis parallelto an optical axis of the second camera, and capturing the first imageset with the first camera may comprise capturing light reflected off amirror extending at a forty-five degree angle relative to the opticalaxis of the first camera.

In some embodiments, rotating the seed to orient the seed parallel tothe first imaginary horizontal line may comprise rotating the seed inresponse to determining the seed is not oriented parallel to the firstimaginary horizontal line. In some embodiments, orienting the seedparallel to the second imaginary horizontal line may comprise orientingthe seed in response to determining the seed is not oriented parallel tothe second imaginary horizontal line, and orienting the seed to positionthe hilum on the secondary imaginary horizontal line may compriseorienting the seed in response to determining the hilum is notpositioned on the second imaginary horizontal line.

In some embodiments, the method may further comprise analyzing a firstimage set corresponding with the projection of the seed onto the firstplane to determine an orientation of the seed relative to the firstimaginary horizontal line, and analyzing a second image setcorresponding with the projection of the seed onto the second plane todetermine an orientation of the seed relative to the second imaginaryhorizontal line.

In some embodiments, analyzing the second image set may compriseidentifying a first longitudinal end and a second longitudinal end ofthe seed, identifying a left rectangular vertical cross section of theseed at the first longitudinal end, identifying a right rectangularvertical cross section of the seed at the second longitudinal end,determining a center of mass of each of the left rectangular verticalcross section and the right rectangular cross section, andinterconnecting the centers of mass of the left rectangular verticalcross section and the right rectangular cross section with an imaginaryline segment. In some embodiments, orienting the seed parallel to thesecond imaginary horizontal line may comprise orienting the seed suchthat the line segment is parallel to the second imaginary horizontalline.

In some embodiments, each of the left rectangular vertical cross sectionand the right rectangular vertical cross section may have a horizontalwidth equal to at least ten image pixels.

In some embodiments, analyzing the second image set may further comprisedetermining an angle of the line segment relative to the secondimaginary horizontal line, and an amount of rotation of the seed toorient the line segment parallel to the second imaginary horizontal lineis based on the determined angle.

In some embodiments, the method may further comprise projecting the seedonto the second plane in response to orienting the seed parallel to thesecond imaginary horizontal line, and analyzing a third image setcorresponding with the projection of the seed onto the second plane inresponse to orienting the seed parallel to the second imaginaryhorizontal line to identify the distance between the hilum and thesecond imaginary horizontal line.

In some embodiments, analyzing the third image set may compriseidentifying a longitudinal end of the seed, determining a center of massof each of the longitudinal end and the hilum, and interconnecting thecenters of mass of the longitudinal end and the hilum with an imaginaryline segment. Additionally, in some embodiments, orienting the seed toposition the hilum on the second imaginary horizontal line may compriseorienting the seed such that the line segment is coincident with thesecond imaginary horizontal line.

In some embodiments, analyzing the third image set may further comprisedetermining an angle of the line segment relative to the secondimaginary horizontal line, and an amount of movement of the seed toorient the line segment coincident with the second imaginary horizontalline is based on the determined angle.

In some embodiments, the method may further comprise determining aheight of the seed for positioning a cutting blade based on theprojection of the seed onto the second plane. The height may be a widthof the seed in a direction perpendicular to the second imaginaryhorizontal line. In some embodiments, the method may further includeattaching the seed to the robotic arm via a suction force.

According to another aspect, a method for imaging a seed includescapturing a plurality of images of a seed, determining an orientation ofthe seed and a location of the hilum of the seed based on the pluralityof captured images, and moving the seed with a robotic arm to orient theseed in a position based on the determined orientation of the seed andthe location of the hilum.

In some embodiments, capturing the plurality of images may comprisecapturing a first image set of the seed with a first camera from a firstperspective, and capturing a second image set of the seed with a secondcamera from a second perspective perpendicular to the first perspective.

In some embodiments, determining the orientation of the seed maycomprise determining an orientation of the seed relative to a firstborder line of the first captured image set, and determining anorientation of the seed relative to a second border line of the secondcaptured image set.

According to another aspect, a seed imaging apparatus includes a roboticarm, one or more light sources, a hollow body having a center axis andconfigured to be lighted by the one or more light sources, a firstcamera configured to capture a first image set of a seed positionedwithin the hollow body. The first image set is captured from a firstperspective along the center axis. The seed imagining apparatus includesa second camera configured to capture a second image set of the seedfrom a second perspective along a second axis perpendicular to thecenter axis, and an electronic controller configured to analyze thefirst image set and the second image set to determine a properorientation of the seed for bisection and to instruct the robotic arm tomove the seed into the proper orientation.

In some embodiments, an optical axis of the first camera may be parallelto an optical axis of the second camera, and the first camera may beconfigured to capture light reflected off a mirror that extends at aforty-five degree angle relative to the optical axis of the firstcamera.

In some embodiments, the robotic arm may be configured to secure theseed by applying a suction force to a side of the seed.

BRIEF DESCRIPTION OF THE DRAWINGS

The detailed description particularly refers to the following figures,in which:

FIG. 1 is a perspective view of a system for preparing seeds for genetransformation;

FIG. 2 is a top plan view of the system of FIG. 1;

FIG. 3 is an exploded perspective view of a portion of a dock of thesystem of FIG. 2;

FIG. 4 is a perspective view of an imaging station of the system of FIG.2;

FIG. 5 is an exploded perspective view of the imaging station of FIG. 4;

FIG. 6 is a perspective view of a cutting device of the system of FIG.2;

FIG. 7 is an exploded perspective view of a cutting block of the cuttingdevice of FIG. 6;

FIG. 8 is a top plan view of the cutting block of FIG. 7;

FIG. 9 is a side elevation view of the cutting block of FIGS. 7-8;

FIG. 10A is a top plan view of the cutting device of FIG. 6 showing thejaws in a disengaged position;

FIG. 10B is a front perspective view of the cutting device of FIG. 6showing the jaws in a disengaged position;

FIG. 11A is a view similar to FIG. 10A showing the jaws in an engagedposition;

FIG. 11B is a view similar to FIG. 10B showing the jaws in an engagedposition;

FIG. 12 is a perspective view of a cutting tool tray of the system ofFIG. 1;

FIG. 13 is a perspective view of a grip assembly of a robotic arm of thesystem of FIG. 1;

FIG. 14 is a simplified block diagram of the system of FIG. 1;

FIGS. 15-16 are block diagrams showing an illustrative operatingprocedure for the system of FIG. 1;

FIGS. 17-19 are block diagrams showing an illustrative procedure fordetermining a desired cutting position and cutting depth for a soybeanseed;

FIGS. 20-26 are illustrations of various preliminary actions in theoperating procedure of FIGS. 15-16, including sterilizing the grips ofthe system of FIG. 1 and selecting a cutting tool;

FIGS. 27-29 are illustrations of an image capture process of theoperating procedure of FIGS. 15-16 to identify a seed to be picked up bythe system of FIG. 1;

FIGS. 30-31 are illustrations of the system of FIG. 1 moving a seed toan imaging station of the system;

FIGS. 32-55 are illustrations of images created during the procedure ofFIGS. 17-19;

FIGS. 56-59 are illustrations of the system of FIG. 1 cutting a seed toprepare the seed for gene transformation;

FIG. 60 is a plan view of a soybean seed;

FIG. 61 is a side elevation view of the soybean seed of FIG. 60;

FIG. 62 is a cross-sectional elevation view of the soybean seed takenalong the line 62-62 in FIG. 60;

FIG. 63 is a cross-sectional elevation view of the soybean seed takenalong the line 63-63 in FIG. 57;

FIG. 64 is a cross-sectional elevation view of the soybean seed takenalong the line 64-64 in FIG. 59; and

FIG. 65 is a plan view of a pair of cotyledon segments prepared usingthe system of FIG. 1.

DETAILED DESCRIPTION OF THE DRAWINGS

While the concepts of the present disclosure are susceptible to variousmodifications and alternative forms, specific exemplary embodimentsthereof have been shown by way of example in the drawings and willherein be described in detail. It should be understood, however, thatthere is no intent to limit the concepts of the present disclosure tothe particular forms disclosed, but on the contrary, the intention is tocover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention as defined by the appended claims.

As used herein, a “cotyledon” may generally refer to an embryonic leafor “primary leaf” of the embryo of a seed plant. A cotyledon is alsoreferred to in the art as a “seed leaf.” Dicotyledonous species, such assoybean, have two cotyledons. A cotyledon segment refers to any portionof a cotyledon, whether it be an entire or whole cotyledon or a fragmentor partial portion of a cotyledon. The “cotyledonary node” refers to thepoint of attachment of the cotyledons to the embyro in the seed orseedling, and may generally refer to the tissue associated with thatpoint of attachment.

As used herein, the term “grasping” refers to holding or seizing thesoybean seed with a tool. Any subsequent mechanism or action that allowsthe soybean seed to be firmly clasped is considered within the scope ofthe term grasping.

As used herein, the term “cutting blade” refers to any cutting tool suchas a razor, knife, water knife, scalpel, chisel, cutter, lance and thelike suitable for cutting or wounding a seed for transformation. In theembodiments disclosed herein, each reference to a cutting blade may besubstituted with a laser or microlaser emission for cutting or woundinga seed for transformation.

As used herein, the term “seed coat” refers to an integument of theovule that serves as a seed's protective coat. Seed coat may bedescribed by the alternative descriptive terms of “testa” or “husk”, inaddition to other similar terms known in the art. Seed coats may containhydrophobic substances such as suberin, cutin, lignin, callose, pectin,waxes, and insoluble products of phenolic oxidation. In legumes, likesoybean, the testa contains a palisade layer of thick-walledmacrosclereid cells, whose caps extend into a suberized sub-cuticle,with a waxy cuticle external to the thicker suberin layer.

As used herein, the terms “embryonic axis” or “embryo axis” refer to themajor portion of the embryo of the plant, and generally includes theepicotyl and hypocotyl.

As used herein, the term “genetically modified” or “transgenic” plantrefers to a plant cell, plant tissue, plant part, plant germplasm, orplant which comprises a preselected DNA sequence which is introducedinto the genome of a plant cell, plant tissue, plant part, plantgermplasm, or plant by transformation.

As used herein, the term “transgenic,” “heterologous,” “introduced,” or“foreign” DNA or gene refer to a recombinant DNA sequence or gene thatdoes not naturally occur in the genome of the plant that is therecipient of the recombinant DNA or gene, or that occurs in therecipient plant at a different location or association in the genomethan in the untransformed plant.

As used herein, the term “explant” refers to a piece of soybean tissuethat is removed or isolated from a donor plant (e.g., from a donorseed), cultured in vitro, and is capable of growth in a suitable media.

As used herein, the term “plant” refers to either a whole plant, planttissue, plant part, including pollen, seeds, or an embryo, plantgermplasm, plant cell, or group of plants. The class of plants that canbe used in the method of the invention is not limited to soybeans, butmay generally include any plants that are amenable to transformationtechniques, including both monocotyledonous and dicotyledonous plants.

As used herein the term “transformation” refers to the transfer andintegration of a nucleic acid or fragment into a host organism,resulting in genetically stable inheritance. Host organisms containingthe transformed nucleic acid fragments are referred to as “transgenic”or “recombinant” or “transformed” organisms. Known methods oftransformation include Agrobacterium tumefaciens or Agrobacteriumrhizogenes mediated transformation, calcium phosphate transformation,polybrene transformation, protoplast fusion, electroporation, ultrasonicmethods (e.g., sonoporation), liposome transformation, microinjection,naked DNA, plasmid vectors, viral vectors, biolistics (microparticlebombardment), silicon carbide WHISKERS™ mediated transformation, aerosolbeaming, or PEG transformation as well as other possible methods.Referring to FIG. 1, a system 10 for preparing seeds or seed explantsfor gene transformation by any known method is shown.

The system 10 is illustratively configured to prepare soybean seeds(hereinafter seeds 12) as part of a transgenic protocol and thedevelopment of transgenic soybean products. Exemplary transgenicprotocols are described in U.S. patent application Ser. No. 14/133,370entitled “IMPROVED SOYBEAN TRANSFORMATION FOR EFFICIENT ANDHIGH-THROUGHPUT TRANSGENIC EVENT PRODUCTION” and U.S. patent applicationSer. No. 14/134,883 entitled “IMPROVED SOYBEAN TRANSFORMATION FOREFFICIENT AND HIGH-THROUGHPUT TRANSGENIC EVENT PRODUCTION,” which areexpressly incorporated herein by reference. It should be appreciatedthat any of the devices and methods described herein can be used inconnection with the transformation methods disclosed in thoseapplications. It should also be appreciated that in other embodimentsany of the devices and methods described herein may be configured foruse with other classes of plants that are amenable to transformationtechniques, including both monocotyledonous and dicotyledonous plants.

The system 10 includes a number of processing stations 14 and a pair ofrobotic arms 16 that move seeds 12 between the processing stations 14.In the illustrative embodiment, each robotic arm 16 is an Epson model C3six-axis articulated arm that is configured to operate independently ofthe other robotic arm. In other embodiments, the robotic arms 16 mayhave a different number of degrees of freedom than those describedherein. For example, the robotic arms 16 may be embodied as robotic armshaving at least independent axes. Each arm 16 includes a grip 18configured to grasp and hold a seed 12. The system 10 may be operatedwith one of the arms 16 out-of service. It should be appreciated that inother embodiments the system may include only a single robotic arm 16 tomove the seeds 12 between the processing stations 14. Additionally, inthe illustrative embodiment, each robotic arm 16 is capable of rotatingthe corresponding grip 18 about its axis by at least 180 degrees.

As shown in FIG. 2, the processing stations 14 and the robotic arms 16are arranged on a table 20. The processing stations 14 include a dock 22positioned at the front of the table 20. The dock 22 includes a pair ofdelivery areas 24 where seeds 12 may be positioned for processing by thesystem 10 and a pair of receiving areas 26 where seeds 12 may bepositioned after processing by the system 10. The stations 14 alsoinclude an imaging station 28 that is operable to capture a number ofimages of the seeds 12. The system 10 also includes a cutting station 30that is operable to cut each seed 12 based on the images captured by thestation 28. The system 10 also includes a sterilization device 32 thatis configured to sterilize each grip 18 of the robotic arms 16 and a binor tray 34 that receives cutting blades for use the cutting station 30.

In use, the system 10 may be operated to cut automatically a number ofsoybean seeds 12 for transformation. To do so, the system 10 may locateone of the seeds 12 on a plate 36 positioned on one of the deliveryareas 24 of the dock 22. The system 10 may then operate the robotic arm16 closest to the plate 36 to grasp the selected seed 12 with the grip18 and move the seed 12 to the imaging station 28. After a series ofimages of the seed 12 are taken, the arm 16 may advance the seed 12 tothe cutting station 30 such that one or more cuts may be made to theseed 12 to prepare it for transformation. After the seed 12 is cut, thearm 16 may move the seed 12 to another plate 38 positioned on one of thereceiving areas 26 of the dock 22. A user may then remove the plate 38including the cut seed to further process the seed in accordance withthe transgenic protocol. Each of these processing steps and the variouscomponents of the system 10 are described in greater detail below inreference to FIGS. 3-59.

Referring now to FIG. 3, a portion of the dock 22 and one of thedelivery areas 24 is shown in greater detail. In the illustrativeembodiment, the other delivery area 24 is identical to the delivery areashown in FIG. 3. The delivery area 24 includes a circular base 40 thatis positioned in an opening defined in a plate 46 of the dock 22. Thebase 40 is sized to receive one of the plates 36, and is constructed ofa transparent material such as, for example, glass, Plexiglas, oracrylic. The base 40 extends from a top surface 42 to a bottom surface(not shown) positioned below the plate 46. Because the base 40 istransparent, objects resting on the top surface 42 of the base 40 arevisible through the bottom surface (i.e., from under the plate 46). Alight-emitting diode (LED) panel 50 is coupled to the bottom of theplate 46 and configured to illuminate the objects resting on the topsurface 42 of the base through the transparent base 40. In theillustrative embodiment, the LED panel emits red light that issufficiently diffuse to minimize reflectance and has variable intensitythat may be controlled by an electronic controller 400 (see FIG. 14), asdescribed in greater detail below.

Each seed carrying plate 36 has a bin 44 defined therein that receivesthe seeds 12. The dock 22 includes a plurality of posts or guide pins 48that surround the circular base 40. As shown in FIG. 3, the pins 48extend upwardly from the plate 46 and are designed to support and/orsecure the plate 36 on the base 40. In other embodiments, the dock 22may include other supporting structure to guide, support, and/or securethe plate 36 on the base 40.

As indicated above, the system 10 is configured to locate the seeds 12on a plate 36 when the plate 36 is positioned on the delivery area 24or, more specifically, when the plate 36 is positioned on the base 40.In the illustrative embodiment, a camera 52 is positioned above thedelivery area 24, as shown in FIG. 1. The camera 52 is electricallycoupled to the electronic controller 400 (see FIG. 14) and is operableto capture images of the plate 36 and seeds 12. As described in greaterdetail below, the images are sent to the controller 400 to determine therelative locations and orientations of the seeds 12 on the plate 36 suchthat the system 10 can direct the robotic arm 16 to the seeds forprocessing. The camera 52 may be embodied as any device suitable forcapturing images, such as a still camera, a video camera, or otherdevice capable of capturing video and/or images. Further, it will beappreciated that an image captured by a camera may be described as aprojection of the scene in the field of view of the camera (e.g., theforeground objects and background) onto a plane perpendicular to theoptical axis of the camera.

Returning to FIG. 2, the dock 22 also includes a pair of receiving areas26 where seeds 12 may be positioned after processing by the system 10.Like the delivery areas 24, each receiving area 26 includes a pluralityof posts or guide pins 48 that define an area sized to receive one ofthe plates 38. Each pin 48 extends upwardly from the plate 46, and thepins 48 cooperate to support and/or secure the plate 38 in the receivingarea 26.

As described above, the system 10 also includes an imaging station 28that is operable to capture a number of images of the seeds 12, whichused to determine the cutting planes for each seed 12. Referring now toFIGS. 4-5, the imaging station 28 includes a lighted dome 54 and twocameras 56, 58 that are secured to the table 20. The camera 56, 58 areelectrically coupled to the electronic controller 400 (see FIG. 14) andare operable to capture images of the inner chamber 62 of the dome 54.In the illustrative embodiment, the lighted dome 54 is an eight-inchdiameter white LED dome light manufactured by Advanced Illumination ofRochester, Vt. The lighted dome 54 includes a concave interior wall 60that defines the bowl-shaped chamber 62 and a circular opening 64 thatpermits access to the chamber 62.

As shown in FIG. 5, the dome 54 also includes a plurality of LEDs 78coupled to the wall 60 to light the chamber 62 during operation. In theillustrative embodiment, the LEDs 78 are formed as a ring ofapproximately 20 LEDs, which are sufficiently diffused to preventreflections onto objects within the dome 54 and may be controlled by thecontroller 400 to vary the intensity of light emitted from the LEDs 78.The ring is mounted around the upper inside edge of the dome 54. Itshould be appreciated that in other embodiments other lighting sourcesmay be used.

The dome 54 includes a convex exterior wall 66 and a plurality of legs68 that extend downwardly from the wall 66 to the table 20. The dome 54has a lower opening 70 extending through the walls 60, 66 at the apex ofthe convex exterior wall 66. In the illustrative embodiment, a centralaxis 72 extends through the centers of the upper opening 64 and thelower opening 70. Another opening 74 extends through the walls 60, 66 onthe side of the dome 54 facing the cameras 56, 58. The opening 74 has alongitudinal axis 76 that extends orthogonal to the central axis 72.

Each of the cameras 56, 58 may be embodied as any device suitable forcapturing images, such as a still camera, a video camera, or otherdevice capable of capturing video and/or images. The cameras 56, 58include optical axes 80, 82, respectively, which are aligned with theopenings 70, 74 of the dome 54. In the illustrative embodiment, theoptical axes 80, 82 are parallel to one another and perpendicular to thecentral axis 72 of the dome 54. As shown in FIGS. 4-5, the longitudinalaxis 76 of the opening 74 is coincident with the axis 82 of the camera58. Additionally, in some embodiments, each of the cameras 56, 58 mayinclude a lens and be positioned such that, in a captured image of aseed 12 positioned within the lighted dome 54, the seed 12 is within atleast half of the field of view of the corresponding camera 56, 58.

The imaging station 28 includes an angled mirror 84 that is positionedbelow the lower opening 70 of the dome 54. The angled mirror 84 isconfigured to reflect light from the chamber 62 toward the camera 56. Inthe illustrative embodiment, the surface 86 of the mirror 84 is angledat a forty-five degree angle relative to each of the central axis 72 andan optical axis 80 of the camera 56. As a result, light from the chamber62 is reflected along the optical axis 80 toward the camera 56. Itshould be appreciated that in other embodiments the mirror may beomitted and the camera 56 positioned directly below the dome 54.Additionally, in other embodiments, the camera 58 may be positionedadjacent to another side of the dome 54. In still other embodiments, oneof the cameras 56, 58 may be omitted.

In the illustrative embodiment, the imaging station 28 includesadditional components to reduce the incidence of stray light enteringthe dome 54 and improve the quality of imaging performed at the imagingstation 28. For example, a cover 90 positioned over the circular opening64 of the dome 54 to reduce the chance that stray light (e.g., from theenvironment of the imaging station 28) enters the dome 54. As shown inFIG. 5, the dome 54 includes a plurality of threaded bores 92 defined inthe rim 94 of the dome 54. Each bore 92 is sized to receive acorresponding fastener 96 to secure the cover 90 to the dome 54.

The cover 90 includes a fabric sheet 100 that is secured to a pad 102.The pad 102 is formed from a high-temperature flexible silicon pad. Inthe illustrative embodiment, the pad 102 is black such that it functionsas a contrasting background to improve the quality of images captured bythe camera 56. It should be appreciated that in other embodiments thepad may be made in another contrasting color. In still otherembodiments, the pad and/or cover may be omitted from the imagingstation 28. As shown in FIG. 5, the cover 90 has a central opening 108that permits the robotic arm 16 to advance a seed 12 into the dome 54.

Another component to improve the quality of imaging is a backstop 106secured to the dome 54. As shown in FIG. 5, the backstop 106 ispositioned within the chamber 62 of the dome 54. The backstop 106, likethe pad 102, is configured to serve as a contrasting background forimages of the seed 12 captured by the camera 58. It should beappreciated that in other embodiments the backstop may be made inanother contrasting color. In still other embodiments, the backstop maybe omitted from the imaging station 28. In yet other embodiments, theimaging station 28 may include an environment for capturing images ofthe seeds 12 in addition or alternatively to the lighted dome 54 suchas, for example, another lighted hollow-bodied structure, a planarmonochromatic backdrop, or some other suitable imaging environment.

As described above, the system 10 also includes a cutting station 30that is operable to cut each seed 12 based on the images captured by thestation 28. Referring now to FIG. 6, the cutting station 30 includes aplatform 110 and a cutting device 112 operable to cut the seed 12 on theplatform 110. The platform 110 includes a pedestal 114 that extendsupwardly from the table 20 and a seed cutting block 116 secured to theupper end 118 of the pedestal 114. The pedestal 114 is formed from ametallic material such as, for example, stainless steel or aluminum. Inthe illustrative embodiment, the cutting block 116 is formed from amagnetic metallic material such as, for example, stainless steel. Itshould be appreciated that in other embodiments the pedestal and/orcutting block may be formed from other rigid materials such as plastics,Teflon, or ceramics.

As shown in FIG. 7, the cutting block 116 is configured to be removedfrom the pedestal 114 for sterilization or repair. In the illustrativeembodiment, the pedestal 114 includes a permanent magnet 120 that ispositioned adjacent to the upper end 118. When the cutting block 116 ispositioned on the pedestal 114, the magnet 120 exerts a force to retainthe cutting block 116 on the pedestal 114. It should be appreciated thatthe magnet is not required to retain the block 116 on the pedestal 114.In the illustrative embodiment, the design of the pedestal 114 issufficient to retain the block 116 thereon.

In the illustrative embodiment, the cutting block 116 has a body 122 anda flange 124 that extends outwardly from the body 122. The lower end 126of the body 122 has a substantially planar bottom surface 128, and thebody 122 has a substantially planar top surface 130. A pair of angledsurfaces 132, 134 extend upwardly from the bottom surface 128. Theangled surface 132 is connected to a back surface 136, which extendsvertically to the top surface 130. As shown in FIG. 7, the angledsurface 132 and the back surface 136 have a slot 138 defined therein.

As shown in FIG. 7, a groove 140 is defined in the upper end 118 of thepedestal 114, and the groove 140 is configured to receive the lower end126 of the block body 122. In the illustrative embodiment, the groove140 is defined by a substantially planar surface 142 and a pair ofangled surfaces 144, 146 that extend upwardly from the surface 142. Inthat way, the configuration of the groove 140 substantially matches theconfiguration of the lower end 126 of the block body 122.

The pedestal 114 also includes a rear wall 148 that faces the backsurface 136 of the cutting block 116 when the block 116 is positioned inthe groove 140. An alignment pin 150 extends outwardly from the rearwall 148. The alignment pin 150 is sized to be received in the slot 138defined in the block 116 to ensure the cutting block 116 is properlypositioned on the pedestal 114.

As shown in FIGS. 7-8, the flange 124 of the cutting block 116 extendsoutwardly from the body 122 to a front wall 154. The flange 124 includesa substantially planar upper wall 156 and a substantially planar lowerwall 158 that is positioned opposite the upper wall 156. The upper wall156 is sized to receive a soybean seed 12. It should be appreciated thatin other embodiments the upper wall 156 may be resized according to thesize of the seed to be cut.

An opening 160 is defined in the front wall 154. A plurality of innerwalls 162 extend inwardly from the front wall 154 of the flange 124 todefine a slot 164 through each of the wall 156, 158. As shown in FIG. 8,the slot 164 is centered in the flange 124, and extends to a back edge166 positioned between a rear edge 174 of the flange 124 and the frontwall 154. As described in greater detail below, the slot 164 is sized toreceive a cutting blade 170 when the blade is rotated vertically.

As shown in FIG. 9, the block body 122 has a substantially planar sidewall 172 that extends upwardly from the rear edge 174 of the flange 124to an upper edge 176. In the illustrative embodiment, the side wall 172extends orthogonal to the upper wall 156. Another side wall 178 isconnected to the upper edge 176 of the side wall 172. The side wall 178extends obliquely relative to the walls 156, 172 to a top edge 180connected to the top surface 130 of the block 116.

Returning to FIG. 6, the cutting station 30 also includes a cuttingdevice 112 that is operable to cut the seed 12 on the platform 110. Thecutting device 112 includes a support arm 190 configured to receive acutting blade 170 and a drive assembly 192 configured to move thecutting blade 170 during the cutting operation. The drive assembly 192includes a drive stage 194 that is secured to the table 20. The drivestage 194 includes a lower body 196 and an upper body 198 configured toslide relative to the lower body 196 in the direction indicated byarrows 200 in FIG. 6. The drive stage 194 includes a linear driveelectric motor (not shown) that is electrically connected to thecontroller 400 and is operable to move the upper body 198 relative tothe lower body 196. In the illustrative embodiment, the drive stage 194is an Aerotech model ANT95-50-L that has approximately 50 millimeters oftravel.

The drive assembly 192 of the cutting device 112 includes anintermediate drive stage 210 that travels with the drive stage 194. Theintermediate drive stage 210 includes a base 212 that is connected tothe upper body 198 of the drive stage 194. The drive stage 210 alsoincludes a platform 214 that is moveably coupled to the base 212. In theillustrative embodiment, the platform 214 is configured to movevertically in the direction indicated by arrows 216 in FIG. 6. The drivestage 210 also includes a linear drive electric motor (not shown) thatis electrically connected to the controller 400 and is operable to movethe platform 214 relative to the base 212. The drive stage 210 isillustratively embodied as Aerotech model ANT95-3-V, which hasapproximately 3 millimeters of travel.

As shown in FIG. 6, the drive assembly 192 includes a rotational stage220 that travels with the other stages 194, 210. The rotational stage220 includes a main body 222 that is connected to the platform 214 ofthe drive stage 210. The rotational stage 220 also includes a mountingshaft 224 that is pivotally coupled to the main body 222. An axis 226 isdefined by the mounting shaft 224, and the shaft 224 is configured torotate about the axis 226 in the directions indicated by arrows 228. Inthe illustrative embodiment, the rotational stage 220 is connected to asource 230 of compressed air such as, for example, a compressor. Thesource 230 is electrically connected to the controller 400. Whenoperated by the controller 400, the source 230 may advance compressedair to the stage 220 such that the shaft 224 is driven pneumaticallyabout the axis 226. The rotational stage 220 is illustratively embodiedas an EMI Plastics Equipment Swiveling Rotary, type RT25.

The support arm 190 of the cutting device 112 is secured to therotational stage 220. As shown in FIG. 6, the support arm 190 includesan elongated body 240 that has an end 242 secured to the mounting shaft224 of the stage 220. The support arm 190 also includes a pair of jaws244, 246 that are secured to the opposite end 248 of the body 240. Inthe illustrative embodiment, each of the jaws 244, 246 has an end 250that is received in a channel 252 defined in the elongated body 240. Thechannel 252 defines a longitudinal axis 254, and the jaws 244, 246 areconfigured to move along the channel 252 toward and away from eachother. In that way, the jaws 244, 246 may be opened or closed. In theillustrative embodiment, the support arm 190 is connected to a source256 of compressed air. The source 256 is electrically connected to thecontroller 400. When operated by the controller 400, the source 256 mayadvance compressed air to the support arm 190 such that the jaws 244,246 are driven pneumatically along the channel 252. The support arm 190is illustratively embodied as an SMC MHZ2-20C1-M9PZ gripper.

The jaws 244, 246 are configured to receive a cutting blade 170.Referring now to FIGS. 10-11, each cutting blade includes a body 260 anda cutting edge 262 extending the length of the body 260. The cuttingedge 262 is offset from the axis of rotation 226 when the cutting blade170 is secured to the jaws 244, 246. The body 260 also includes a pairof oblong mounting holes 264, which are engaged by the jaws 244, 246 tosecure the cutting blade 170 to the device 112. The cutting blade 170 isillustratively formed from a metallic material such as steel.

Each of the jaws 244, 246 extends from the end 250 to a tip 270. Each ofthe jaws 244, 246 includes an inner tab 272 positioned along an inneredge 274 of the tip 270. Each tab 272 is sized to be positioned in oneof the holes 264 of a cutting blade 170. In the illustrative embodiment,each of the jaws 244, 246 also includes a slot 276 (see FIG. 10B) thatis formed at the base of each tab 272. In the illustrative embodiment,each slot is configured to capture the blade and hold it level. As shownin FIGS. 11A and B, the blade 170 is advanced into the slots 276 whenthe jaws 244, 246 are moved apart, thereby securing the blade to thejaws 244, 246. Each of the jaws 244, 246 also includes an outer tab 278that is positioned along an outer edge 280 of the tip 270. The outer tab278 includes a beveled edge 282 to assist with alignment of the blade170 as it is inserted onto the jaws 244, 246.

As shown in FIG. 11A, the elongated body 240 of the support arm 190 hasa longitudinal axis 284. In the illustrative embodiment, the cuttingblade 170 is offset from the axis 284 when secured to the jaws 244, 246.During operation, the offset of the cutting blade 170 from the axis 284lowers the cutting blade 170 to reduce the risk that the cutting bladewill contact the robotic arm when cutting the soybean seed.

Referring now to FIG. 12, a tray 34 for holding unused cutting blades170 is positioned between the robotic arms 16. The tray 34 includes acontainer 302 positioned above a light source 304. The container 302 isillustratively formed from a transparent material such as, for example,Plexiglas. The container 302 includes a bottom wall 306 and a pluralityof side walls 308 that extend upwardly from the bottom wall 306. Thewalls 306, 308 cooperate to define a chamber 310 sized to receive unusedcutting blades 170.

In the illustrative embodiment, the light source 304 of the tray 34 ispositioned below the bottom wall 306. The light source 304 is operableto project light through the bottom wall 306 into the chamber 310. Thelight source 304 is illustratively embodied as a red light-emittingdiode (LED). It should be appreciated that in other embodiments othercolored LEDs may be used. In still other embodiments, other lightingsources may be used.

The system 10 includes a tray camera 312, which is mounted above thetray 34. The camera 312 is operable to capture images of the contents ofthe chamber 310. The camera 312 is electrically coupled to an electroniccontroller 400 (see FIG. 14). As described in greater detail below, theimages may be sent to the controller 400 to determine the relativelocations and orientations of the blades 170 in the tray 34 such thatthe system 10 can direct the robotic arm 16 to the blades 170 forretrieval.

Referring now to FIG. 13, each robotic arm 16 of the system 10 includesa grip assembly 320 configured to grasp and hold a soybean seed 12. Inthe illustrative embodiment, the grip assembly 320 includes a body 322that is attached to a distal section 324 of each arm 16. The gripassembly 320 also includes a suspension mechanism 326 that connects thebody 322 to a grip 18. The body 322 has a proximal disk 328 that issecured to the distal arm section 324 and a plurality of posts 330 thatextend from the disk 328 to a distal disk 332.

The suspension mechanism 326 extends from a proximal end 334 that issecured to the disk 332 to a distal end 336. As shown in FIG. 13, thegrip 18 is secured to the distal end 336 of the suspension mechanism326. The suspension mechanism 326 is configured to permit some axialmovement of the grip 18, as indicated by arrows 338, 340, such that thegrip 18 may be advanced into contact with a soybean seed 12 withoutcrushing the seed. In the illustrative embodiment, the suspensionmechanism 326 includes a biasing element such as, for example, a helicalspring 342, that biases the grip 18 outward, in the direction indicatedby arrow 340.

The grip 18 of the assembly 320 is configured to grasp and hold a seed12. In the illustrative embodiment, the grip 18 includes a cylindricalbody 350 that is secured to the distal end 336 of the suspensionmechanism 326. The body 350 is formed from an elastomeric material suchas, for example, Viton, which is commercially available from DuPontCorporation. It should be appreciated that in other embodiments otherelastomeric materials may be used. The body 350 includes a bellows,which provides the body 350 with limited flexibility. The body 350 alsohas a high temperature rating to permit sterilization of the grip 18. Inthe illustrative embodiment, the temperature rating is 446 degreesFahrenheit. It should be appreciated that in other embodiments otherelastomeric materials may be used.

The grip assembly 320 is configured to grasp and hold the seed 12 viavacuum. To do so, the grip 18 includes a hollow passageway 352 thatextends longitudinally through the body 350 along an axis 358. Thepassageway 352 is connected to passageways 354 defined in the suspensionmechanism 326 and the body 322 of the grip assembly 320 and a negativepressure source 356. The negative pressure source 356 is illustrativelyembodied as a pump and is electrically coupled to the controller 400.The controller 400 may operate the source 356 to draw a vacuum throughthe passageways 352, 354 and secure a seed 12 to the grip 18. In theillustrative embodiment, the grip 18 has a radius of less than fiftypercent of the average length of a seed 12, which may vary depending on,for example, the particular species of the seed 12.

As shown in FIG. 13, the grip assembly 320 also includes a secondarycover 360 that is secured to the body 350. The secondary cover 360 isdesigned to prevent stray light from entering the lighted dome 54 duringimaging of the seed 12. The cover 360 includes a bottom pad 362 formedfrom a black foam material and a top pad 364 that is formed from blackfelt. In the illustrative embodiment, the cover 360 is secured to thedistal disk 332 via adhesive. It should be appreciated that in otherembodiments the cover 360 may be secured with fasteners such as screwsor bolts. The cover 360 has a diameter of approximately 3.5 inches,which is sufficient to enclose central opening 108 of the cover 90 ofthe dome 54.

Referring now to FIG. 14, the system 10 includes an electroniccontroller 400. The controller 400 is, in essence, the master computerresponsible for interpreting electrical signals sent by sensorsassociated with the system 10 and for activating or energizingelectronically-controlled components associated with the system 10. Forexample, the electronic controller 400 is configured to control theoperation of the cameras 52, 56, 58, 312, dome light 78, robotic arms16, drive stages 194, 210, and so forth. While the electronic controller400 is shown as a single unit in FIG. 14, the controller 400 may includea number of individual controllers for the various components as well asa central computer that sends and receives signals from the variousindividual controllers. The electronic controller 400 also determineswhen various operations of the system 10 should be performed. As will bedescribed in more detail below, the electronic controller 400 isoperable to control the components of the system 10 such that the system10 selects and processes soybean seeds 12 for use in transgenicprotocols.

To do so, the electronic controller 400 includes a number of electroniccomponents commonly associated with electronic units utilized in thecontrol of electromechanical systems. For example, the electroniccontroller 400 may include, amongst other components customarilyincluded in such devices, a processor such as a microprocessor 402 and amemory device 404 such as a programmable read-only memory device(“PROM”) including erasable PROM's (EPROM's or EEPROM's). The memorydevice 404 is provided to store, amongst other things, instructions inthe form of, for example, a software routine (or routines) which, whenexecuted by the microprocessor 402, allows the electronic controller 400to control operation of the system 10.

The electronic controller 400 also includes an analog interface circuit406. The analog interface circuit 406 converts the output signals fromthe various components into signals that are suitable for presentationto an input of the microprocessor 402. In particular, the analoginterface circuit 406, by use of an analog-to-digital (A/D) converter(not shown) or the like, converts the analog signals generated by thesensors into digital signals for use by the microprocessor 402. Itshould be appreciated that the A/D converter may be embodied as adiscrete device or number of devices, or may be integrated into themicroprocessor 402. It should also be appreciated that if any one ormore of the sensors associated with the system 10 generate a digitaloutput signal, the analog interface circuit 406 may be bypassed.

Similarly, the analog interface circuit 406 converts signals from themicroprocessor 402 into output signals which are suitable forpresentation to the electrically-controlled components associated withthe system 10 (e.g., the robotic arms 16). In particular, the analoginterface circuit 406, by use of a digital-to-analog (D/A) converter(not shown) or the like, converts the digital signals generated by themicroprocessor 402 into analog signals for use by theelectronically-controlled components associated with the system 10. Itshould be appreciated that, similar to the A/D converter describedabove, the D/A converter may be embodied as a discrete device or numberof devices, or may be integrated into the microprocessor 402. It shouldalso be appreciated that if any one or more of theelectronically-controlled components associated with the system 10operate on a digital input signal, the analog interface circuit 406 maybe bypassed.

Thus, the electronic controller 400 may operate to control the operationof the system 10. In particular, the electronic controller 400 executesa routine including, amongst other things, a control scheme in which theelectronic controller 400 monitors the outputs of the sensors associatedwith the system 10 and controls the inputs to theelectronically-controlled components of the system 10. To do so, theelectronic controller 400 performs numerous calculations, eithercontinuously or intermittently, including looking up values inpreprogrammed tables, in order to execute algorithms to perform suchfunctions as energizing the robotic arms 16, activating the cameras 52,56, 58, 312, energizing the drive stages 194, 210, varying the lightintensity of the LEDs 78 and LED panel 50 to improve image contrast, andso on.

In operation, the system 10 may be operated in accordance with theexemplary procedure outlined in FIGS. 15-19 to automatically select andprocess soybean seeds 12 for use in a transgenic protocol. For example,the soybean may be prepared by splitting the cotyledons of a seed 12along the hilum to separate the cotyledons. Removal of a portion of theembryonic axis leaves part of the axis attached to the cotyledons priorto transformation. The removal of the embryonic axis may be made bytrimming of the embryonic axis with the cutting device 112. Typically,between ⅓ and ½ of the embryo axis is left attached at the nodal end ofthe cotyledon

As shown in FIGS. 20-29, the system 10 engages in preliminary steps tosterilize the grips 18 of the robotic arms 16, select a cutting blade170 for the cutting station 30, and capture images of the seeds 12located in the delivery areas 24. Thereafter, the system 10 operates oneof the robotic arms 16 to pick-up a seed 12 from one of the deliveryareas 24 and advance the seed 12 to the imaging station 28, as shown inFIGS. 27-31. A number of images may be captured by the imaging station28, as shown in FIGS. 32-55, before the seed 12 is advanced to thecutting station 30. As shown in FIGS. 56-59, the cutting station 30 maybe operated to make one or more cuts to the seed 12 to prepare it fortransformation. The cut seed may then be advanced to one of thereceiving areas 26. A user may then remove the seed from the system 10for further processing. The system 10 may then engage in a number ofcleaning and maintenance tasks before picking up and processing anotherseed 12.

As shown in FIG. 60-62, a soybean seed 12 includes a pair of cotyledons412, 414, which are encased in a seed coat 416. The soybean seed 12 hasa longitudinal axis 418, which is defined along its maximum dimension,and extends through opposite longitudinal ends 420, 422 of the soybeanseed 12. As shown in FIG. 60, the axis 418 extends between thecotyledons 412, 414.

The soybean seed 12 also includes a hilum 424 positioned between theends 420, 422 of the soybean seed 12. In the illustrative embodiment,the hilum 424 includes an outer section 426 that is positioned outsideof the seed coat 416 and an inner section 428 that is positioned underthe seed coat 416.

As shown in FIGS. 60-61, the hilum 424 is dorsally located above thecotyledons 412, 414. The outer section 426 of the hilum 424 ispositioned on a dorsal side 430 of the soybean seed 12. The hilum 424may also be viewed from the lateral side 432 (see FIG. 61) or the medialside 434 (see FIG. 5) of the soybean seed 12. As shown in FIG. 61, thehilum 424 has a longitudinal axis 436 that extends parallel to theoverall longitudinal axis 418 of the seed 12. As shown in FIG. 60, thelongitudinal axis 436 lies in a common plane 438 with the axis 418 ofthe seed 12.

An embryonic axis 440 of the soybean seed 12 connects the cotyledon 412to the cotyledon 414. The embryonic axis 440 is encased with thecotyledons 412, 414 in the seed coat 416. As shown in FIG. 60, theembryonic axis 440, like the hilum 424, is centered on the longitudinalaxis 418 of the soybean seed 12. As shown in FIG. 62, the embryonic axis440 extends from a tip 442 positioned above the inner section 428 of thehilum 424 to a base 444 positioned adjacent to the longitudinal end 420of the seed 12. It should be appreciated that in other embodiments theembryonic axis 440 may not overlap with the hilum 424 such that the axistip 442 is spaced apart from the inner section 428 of the hilum.

Referring to FIG. 62, the internal structure of the soybean seed 12 isshown in greater detail. The seed coat 416 includes a thin outer layer450 that surrounds the cotyledons 412, 414 and the embryonic axis 440.The inner section 428 of the hilum 424 is attached to the underside ofthe layer 450, while the outer section 426 of the hilum 424 is connectedto an edge 452 of the outer layer 450. The embryonic axis 440 extendsaround a portion of the outer circumference of the seed 12 from its tip442 to its base 444 positioned adjacent to the seed end 420

Referring now to FIGS. 15-16, an illustrative operating procedure 1000for preparing the soybean seed 12 for transformation with the system 10is shown. It will be appreciated that prior to commencement of theprocedure 1000, the controller 400 may calibrate the system 10, providemessages to the user, retrieve user input, initialize safety mechanisms(e.g., a light curtain), and perform other setup functions. For example,if not done already, the controller 400 may calibrate the system 10using any suitable protocol to map or otherwise correlate the coordinatesystem of the robotic arms 16 to the coordinate systems of the variouscameras 52, 56, 58, 312 such that locations of objects captured inimages may be translated to a location of that object relative to thearms 16. Further, the controller 400 may provide setup instructions tothe user on a display 460 (e.g., to place the plate 36 on the deliveryareas 24), retrieve input from the user via a user input device 462(e.g., a desired trim depth of the embryonic node of the seeds 12, abisecting depth of the seeds 12, etc.). The user input device 462 may beembodied as any integrated or peripheral device such as a keyboard,mouse, touchscreen, and/or other input devices configured to perform thefunctions described herein.

In block 1002, the system 10 sterilizes the grips 18 of the robotic arms16. To do so, the controller 400 operates each robotic arm 16 to insertits corresponding grip 18 into a container filled with ethanol oranother suitable sterilizing solution. The solution illustrativelycontains 70% alcohol. The robotic arm 16 may be operated to move thegrip 18 up and down and side to side within the ethanol for some periodof time before advancing the grip 18 into a sterilizer 32, as shown inFIG. 20. In the illustrative embodiment, each sterilizer 32 is a dryglass bead sterilizer such as, for example, an InoTech BioScience Steri250. The robotic arm 16 may again be operated to move the grip 18 up anddown within the sterilizer 32 for a few seconds in the illustrativeembodiment. The arm 16 may then withdraw the grip 18 from the sterilizer32 such that the grip 18 is permitted to cool.

Due to the heat generated by the sterilizer 32, the bellows of the grip18 may become stuck together such that performance of the grip 18 may beimpaired. To separate the bellows, the robotic arm 16 may then move thegrip 18 into contact with a flat sterile surface, such as, for example,the top surface 130 of the cutting block 116, as shown in FIG. 21. Thecontroller 400 may then activate the negative pressure source 356 toseal the grip 18 to the cutting block 116. As shown in FIGS. 22-23, thegrip 18 is moved away from the cutting block 116 in 1 mm incrementsuntil the suction is broken.

Returning to FIG. 15, the procedure 1000 may then advance to block 1004.In block 1004, a cutting blade 170 is selected and retrieved from thetray 34. To do so, the controller 400 operates the camera 312 to captureimages of the blades 170 in the tray 34. One such image 500 is shown inFIG. 24. As shown in FIGS. 24-25, the blades 170 may be positioned inarbitrary locations and orientations relative to one another within thetray 34. The controller 400 may process the captured image 500 toidentify the location 516 of one of the blades 170 in the tray 34, whichmay be reflected by an analyzed image 518 as shown in FIG. 25.

For example, in the illustrative embodiment, the controller 400 utilizesa geometric object-identifying function of the software package includedwith the Epson model C3 six-axis articulated arms. In particular, ablade reference image (not shown) loaded by the user and stored in thememory device 404 of the controller 400 is compared to the capturedimage 500 of the blades 170 to identify a match 502. The geometricobject-identifying function employs an algorithmic approach thatidentifies matches to a reference image (i.e., an object model) by usingedge-based geometric features. Further, the geometric object-identifyingfunction includes various parameters such as a reference image to beused for comparison to another image and an acceptance or tolerancelevel required for the match 502. The acceptance level corresponds witha likelihood of a match 502 and may, without loss of generality, beconsidered herein as a normalized value between 0 and 1. Accordingly, ifthe acceptance level is set to 0.5, only those objects in the analyzedimage having at least a fifty percent likelihood of a match 502 with thereference image based on a suitable imaging algorithm will be identifiedby the controller 400. In a specific embodiment, the acceptance levelmay correspond with a percentage of a reference image that must beidentified in a continuous region of an analyzed image to constitute amatch 502.

In the illustrative embodiment, the controller 400 analyzes the capturedimage 500 using the matching algorithm, the blade reference image, and anormalized acceptance level of 0.4 (i.e., 400 out of 1000) to determinewhether there are any blades 170 on the tray 34. An assumption is madethat if any blades 170 are on the tray 34, even if the blades 170 areoverlapping, such an acceptance level should return the identifiedlocations of those blades 170 on the tray 34. As such, in anotherembodiment, a different acceptance level may be used. If no blades 170are identified, the controller 400 determines that no blades 170 arelocated on the tray 34 and processes the error. For example, thecontroller 400 may instruct the user of the system 10 via a display 460to place additional blades 170 on the tray 34 or otherwise remedy theerror.

If the controller 400 determines that at least one blade 170 is locatedon the tray 34, the controller 400 analyzes the captured image 500 againwith the acceptance level set to a higher threshold value such as 0.95(i.e., 950 out of 1000) to identify a blade 170 that does not overlapwith another blade 170 on the tray 34. If at least one non-overlappingblade 170 is identified, the controller 400 selects that blade 170 foruse. However, if non-overlapping blades 170 are identified, thecontroller 400 executes a protocol to separate the overlapping blades170.

In doing so, the controller 400 identifies the location of a blade 170that overlaps with another blade 170 on the tray 34. For example, thecontroller 400 may use the image locations identified with thenormalized acceptance level set at 0.4, if saved, or similarly analyzethe image 500. When the group of blades 170 has been identified, thecontroller 400 determines the geometric center of the group using asuitable imaging algorithm (e.g., by detecting a center of mass of thegroup) and instructs the corresponding robotic arm 16 to move the gripassembly 320 into position for grasping the group of blades 170 at theidentified center of mass.

To grasp an object from the tray 34 or plate 36, the grip assembly 320is positioned above a grip location or point 504 of the object such thatthe hollow passageway 352 of the grip assembly is approximatelycollinear with the point 504. The grip assembly 320 is then advanceddownward toward the object until the grip 18 is in full contact with theouter surface of the object. As described above, the suspensionmechanism 326 operates to prevent the object from being crushed whileensuring that the grip 18 is in full contact with the object's surfaceto provide limited loss of suction. The negative pressure source 356 maythen be activated to secure the object to the grip 18.

Similarly, if there are no non-overlapping blades, the grip assembly 320may grasp a group of blades 170 at the identified center of mass. Thecontroller 400 may then operate the arm 16 to move the grip assembly 320vertically a short distance (e.g., one inch) above the surface of thetray 34 and horizontally a short distance but still within a perimeterof the tray 34. The controller 400 may then deactivate the negativepressure source 356 to drop the group of blades 170 back onto the tray34. It will be appreciated that one or more of the blades 170 within thegroup may fall during transport. The controller 400 operates the camera312 to capture another image of the blades 170 in the tray 34 andanalyzes the new image similarly to that described above to identify anon-overlapping blade 170. If no non-overlapping blade 170 isidentified, the controller 400 may again instruct the grip assembly 320to grasp a group of blades 170 and drop the blades 170 on anotherlocation within the tray 34. The controller 400 may continue to repeatthe routine until a non-overlapping blade 170 is identified selected foruse.

In another embodiment, the controller 400 may implement other proceduresfor separating overlapping blades 170 and identifying a particular blade170 for selection. Further, the controller 400 may utilize any suitableimage processing algorithms and techniques to identify the locations ofthe blades 170 in the tray 34. For example, the controller 400 mayutilize feature detection algorithms, techniques, and filters such asSpeeded Up Robust Features (SURF), Scale-Invariant Feature Transform(SIFT), Multi-Scale Oriented Patches (MOPS), Canny, image gradientoperators, and Sobel filters to identify features (e.g., interest pointssuch as corners, edges, blobs, etc.) of the image 500 and the bladereference image. In some embodiments, the controller 400 may utilizefeature matching algorithms such as the Random Sample Consensus (RANSAC)algorithm to determine whether any features identified in the image 500and the blade reference image correspond with one another and, if so,the corresponding locations of those features. Additionally oralternatively, the controller 400 may utilize image segmentationalgorithms (e.g., pyramid segmentation, watershed algorithms, etc.) foridentifying objects in an image. It will be appreciated that, dependingon the particular embodiment, the controller 400 may utilize any one ormore of the algorithms described above during the analyses of capturedimages.

After the controller 400 has identified a blade 170, the controller 400uses blade features such as, for example, the mounting holes 264 of theblade 170 to locate the cutting edge 262 of the blade. The controller400 may then calculate the rotation angle of the blade 170 with respectto the grip assembly 320 and calculate the correct position point 504 onthe blade for attachment of the grip 18. The grip assembly 320 graspsthe blade at the point 504 in a similar manner to that described above.

Returning to FIG. 15, the procedure 1000 advances to block 1006 once thegrip 18 has picked up a blade 170. In block 1006, the controller 400operates the robotic arm 16 and the cutting device 112 to secure thecutting blade 170 to the cutting device 112. To do so, the controller400 activates the robotic arm 16 to move the cutting blade 170 to thecutting station 30 and position the cutting blade 170 above the jaws244, 246 of the cutting device 112. To position the cutting blade 170 onthe jaws 244, 246, the robotic arm 16 may be moved in a circular motionto align the oblong mounting holes 264 of the blade 170 with the tabs272 of the jaws 244, 246. The beveled edges 282 of the outer tabs 278assist in guiding the blade 170 onto the tabs 272. When the blade 170 ispositioned on the tabs 272, the grip 18 is moved downward, causing theblade 170 to deflect slightly. As shown in FIG. 26, the controller 400may then operate the jaws 244, 246 to secure the blade 170 to thecutting device 112. A camera (not shown) may be used to capture imagesof the blade 170 positioned on the cutting device 112, and thecontroller 400 may use image processing techniques similar to thosedescribed above confirm the blade 170 is properly positioned on the jaws244, 246.

With the blade 170 positioned on the jaws 244, 246, the controller 400may operate the compressed air source 256 to move the jaws 244, 246outward along the channel 252 of the elongated body 240. As the jaws244, 246 are advanced outwardly, portions of the cutting blade 170 areadvanced into the slots 276 formed at the base of the tabs 272, therebysecuring the cutting blade 170 to the jaws 244, 246. The controller 400may deactivate the vacuum source 356 to release the cutting blade 170from the grip 18 and operate the robotic arm 16 to move the grip 18 outof the cutting station 30.

Returning to FIG. 15, the procedure 1000 advances to block 1008 in whichthe controller 400 operates the camera 52 to capture images of the seeds12 on a plate 36 positioned in the corresponding delivery area 24. Onesuch image 510 is shown in FIG. 27. As shown in FIG. 27 and similar tothat described above with regard to the blades 170 in the tray 34, theseeds 12 may be arbitrarily positioned relative to one another withinthe plate 36.

In block 1010, the controller 400 may process the captured image 510 todetermine the location of one of the seeds 12 on the plate 36 forselection. To do so, the controller 400 may analyze the captured image510 using a matching algorithm (e.g., the geometric object-identifyingfunction described above) to compare a reference image 512 of a seed 12lying on its side, as shown in FIG. 28, to the captured image 510. Inthe illustrative embodiment, the controller 400 assumes that the user ofthe system 10 has placed each of the seeds 12 on its side within theplate 36 in a single layer. Accordingly, there is a high likelihood ofdetecting a match 522. However, in other embodiments, the controller 400may not make such an assumption; rather, the controller 400 may, forexample, determine which seeds 12, if any, are not appropriatelyoriented and ignore those seeds 12. The system 10 may generate a warningor instruct the user to remedy the situation (e.g., via the display460), or otherwise handle the error. In other embodiments, thecontroller 400 may use blob detection or other image analysis algorithmsto determine the location of the seeds 12 on the plate 36.

In any case, the controller 400 determines the locations 520 of one ormore seeds 12 on the plate 36, which may be reflected by an analyzedimage 514 as shown in FIG. 29. Further, in some embodiments, thecontroller 400 determines an angle of rotation of the identified seed(s)12 on the plate 36 relative to the seed 12 depicted in the referenceimage 512. Based on that information, the controller 400 may determinean amount by which to rotate the grip 18 to place the secured seed 12 ina predefined orientation (e.g., zero degree angle relative to thecoordinate system of the robotic arm 16) on the grip 18. By doing so,the controller 400 may be able to identify the hilum and embryo axis(i.e., the embryonic axis) of the seed 12 as described below and saveprocessing time.

Returning to FIG. 15, the procedure 1000 advances to block 1012. Inblock 1012, the controller 400 identifies and selects (e.g., arbitraryor algorithmically) one of the seeds for trimming and bisection by thesystem 10. In the illustrative embodiment, the controller 400 identifiesthe center of mass of the selected seed 12 and uses that as the point504 to attach the grip 18 as shown in FIG. 30. In block 1014, the gripassembly 320 grasps the selected seed 12 at its center of mass. To doso, the grip assembly 320 is positioned above the center of mass (i.e.,the point 504) such that the hollow passageway 352 of the grip assemblyis approximately collinear with the point 504. The grip assembly 320 isthen advanced downward toward the selected seed until the grip 18 is infull contact with the outer surface of the seed. As described above, thesuspension mechanism 326 operates to prevent the seed from being crushedwhile ensuring that the grip 18 is in full contact with the seed'ssurface to provide limited loss of suction. The negative pressure source356 may then be activated to secure the seed to the grip 18. Theprocedure 1000 may then advance to block 1016 in which the robotic arm16 moves the gripped seed 12 through the central opening 108 in thecover 90 and into the chamber 62 of the lighted dome 54, as shown inFIG. 31. In the illustrative embodiment, the gripped seed 12 ispositioned within the chamber 62 at a location that is within the fieldsof view of each of the cameras 56, 58 (e.g., an intersection point ofthe optical axes 80, 82). For example, in some embodiments, the grippedseed 12 is positioned, at least in part, within the focal plane of eachof the cameras 56, 58.

When the seed 12 is positioned in the chamber 62 of the lighted dome 54,the procedure advances to block 1018, as shown in FIG. 15. In block1018, the controller 400 determines the proper orientations of thegripped seed 12 for trimming and bisecting the seed 12 with the cuttingdevice 112. That is, the controller 400 determines how the seed 12 ispositioned relative to the grip 18 so that the robotic arm 16 canproperly position the seed 12 on the cutting block 116 for trimming andbisecting the embryo of the seed 12. To do so, an illustrative operatingprocedure 1200, as shown in FIG. 17, may be used. Although the procedure1200 is described herein with regard to analyzing several still imagesin a linear manner, it will be appreciated that, in some embodiments,the controller 400 may perform multiple image analyses in parallel orcontinuously analyze video, for example.

The procedure 1200 may begin with block 1202 in which the controller 400operates the camera 58 to capture an image 530 of the gripped seed 12from a side perspective. In block 1204, the controller 400 analyzes theimage 530 to determine whether a hilum 424 of the seed 12 is visible onthe seed 12. That is, the controller 400 determines whether the hilum424 (see FIG. 32) is within a field of view of the camera 58. To do so,the controller 400 may utilize any suitable image processing algorithmssuch as those described herein. For example, in the illustrativeembodiment, the controller 400 utilizes a correlation model that usesshadows (e.g., grayscale pixel intensity) to model the seed 12 andidentify a match 534, if any, between the seed 12 and a reference image536 of a seed hilum as shown in FIG. 32. In particular, the correlationmodel performs a pixel-to-pixel match of the reference image 536 to thecaptured image 530.

In block 1206, the controller 400 determines whether the hilum 424 ofthe seed 12 is within the field of view of the camera 58. If so, theprocedure 1200 advances to block 1210. However, if the controller 400determines that the hilum 424 is not within the field of view of thecamera 58, the procedure advances to block 1208.

In block 1208, the controller 400 may reorient the seed 12 such that thehilum is within the field of view of the camera 58. In particular, thecontroller 400 operates the robotic arm 16 to rotate the seed 12 aboutthe axis 358 of the grip 18 until the hilum 424 is within the field ofview of the camera 58. In some embodiments, the robotic arm 16 rotatesthe seed 12 by an incremental angle, the camera 58 captures a new imageof the gripped seed 12, and the controller 400 analyzes the new image todetermine whether the hilum 424 is now within the field of view of thecamera 58. If not, the routine may be repeated until the hilum 424 iswithin the field of view of the camera 58. In an embodiment, the roboticarm 16 may first rotate the seed 12 by an angle of 180 degrees toexpedite the process of locating the hilum 424. Once the hilum 424 isdetermined to be within the field of view of the camera 58, theprocedure 1200 may advance to block 1210.

In block 1210, the controller 400 operates the camera 56 to capture animage 540 of the gripped seed 12 from a bottom perspective as shown inFIG. 33. The procedure 1200 then advances to block 1212 of FIG. 18. Inblock 1212, the controller 400 analyzes the captured image 540 toidentify a longitudinal axis 542 (i.e., a major axis) of the seed 12 inthe image 540. In the illustrative embodiment, the controller 400utilizes a blob detection algorithm to locate the seed 12 in thecaptured image 540 and determine the principal axes (i.e., the majoraxis and the minor axes) of the seed 12. For example, the blob detectionalgorithm may identify the seed 12 in the captured image 540 as a blob,determine the center of mass and edges of that blob, and approximate themajor and minor axes based on that information.

It will be appreciated that the particular blob detection algorithmutilized may vary depending on the particular embodiment. For example,in the illustrative embodiment, the controller 400 uses blob detectionalgorithms of the software package included with the Epson model C3six-axis articulated arms. In some embodiments, the blob detectionalgorithms may be based on Difference of Gaussian (DoG), Laplacian ofGaussian (LoG), Hessian determinants, and/or other operators. In anembodiment, the controller 400 may utilize one or more of the blobdetection algorithms described in, for example, Lindeberg, DetectingSalient Blob-Like Image Structures and Their Scales with a Scale-SpacePrimal Sketch: A Method for Focus-of-Attention, 11(3) InternationalJournal of Computer Vision, 283-318 (1993). Further, in someembodiments, the controller 400 may draw a rectangular border 548 aroundthe seed 12 (or other objects) in a processed version of the capturedimage 540 to indicate the location of the identified seed 12 (or otherobjects). In other embodiments, the controller 400 may utilize otherimage analysis algorithms (e.g., image segmentation) to identify theseed 12 and/or the longitudinal axis 542.

The controller 400 further determines an angle 544 of rotation of themajor or longitudinal axis 542 of the seed 12 relative to a horizontalaxis 546 or other horizontal line 554 of the captured image 540. Inother words, the angle 544 defined between the longitudinal axis 542 andthe horizontal axis 546 or other horizontal line 554 is determined. Inthe illustrative embodiment, the camera 56 is configured to capturerectilinear images; as such, the horizontal axis 546 of the capturedimage 540 may be considered parallel to an edge of the camera 56.

As shown in FIG. 34, the robotic arm 16 is capable of reorienting thegripped seed 12 within the lighted dome 54. For example, depending onthe reorientation necessary, the robotic arm 16 may change theorientation of the seed 12 by rotating and/or translating the seed 12.Accordingly, in block 1214 of FIG. 18, the controller 400 operates therobotic arm 16 to orient the seed 12 such that the longitudinal axis 542is parallel to the horizontal axis 546 as shown in FIG. 35. Inparticular, the robotic arm 16 rotates the seed 12 about the axis 358.In some embodiments, the controller 400 may not require preciseparallelism but may establish a tolerance for the angle 544. In someembodiments, the tolerance may be less than or equal to 1.0 degree. Inother embodiments, the tolerance may be less than or equal to 0.5degrees. In still other embodiments, the tolerance may be less than orequal to 0.3 degrees for the angle 544. It should be appreciated thatsimilar tolerances may be established for any of the measurementsdescribed herein. As indicated above, the camera 56 and the robotic arm16 are calibrated such that their coordinate systems are mapped to oneanother, so orienting the seed 12 in such a way effectively aligns thelongitudinal axis 542 of the seed 12 with an axis of the robotic arm'scoordinate system.

Returning to FIG. 18, the procedure 1200 may advance to block 1216 inwhich the controller 400 operates the camera 58 to capture an image 550of the gripped seed 12. As shown in FIG. 36, the image 550 is a sideelevation view of the seed from the perspective of the camera 58. Theimage 550 may be analyzed in block 1218 to identify the gripped seed 12and a longitudinal axis 552 of the seed 12. It will be appreciated thatthe longitudinal axes 552, 578 may or may not be coincident with oneanother due to the irregular shape of the seed 12. The controller 400may utilize a blob detection algorithm to identify a location 556 of theseed 12 and/or locate the longitudinal axis 552 in the captured image550 in a similar manner to that described above with regard to theanalysis of the captured image 540.

In the illustrative embodiment, the controller 400 identifies a leftvertical slice 560 or cross section of the seed 12 at a leftlongitudinal end 562 of the seed 12 and a right vertical slice 564 orcross section of the seed 12 at a right longitudinal end 566 of the seed12 in the captured image 550. As shown in FIG. 37, each of the verticalslices 560, 564 is at least one pixel in width. In the illustrativeembodiment, the width of the slices is 25 pixels, but the width may varyin other embodiments. The controller 400 determines a center of mass 570of the left vertical slice 560 of the seed 12 and a center of mass 572of the right vertical slice 564 of the seed 12. The longitudinal axis552 of the seed 12 in the captured image 550 is defined as the lineintersecting both centers of mass 570, 572. In other words, thelongitudinal axis 552 runs through the centers of mass of thelongitudinal ends 562, 566 of the seed 12. The controller 400 furtherdetermines an angle 574 of the longitudinal axis 552 relative to ahorizontal axis 576 or other horizontal line 578 of the captured image550.

The procedure 1200 may advance to block 1220 in which the seed 12 isreoriented. In particular, the controller 400 operates the robotic arm16 to orient the seed 12 such that the longitudinal axis 552 is parallelto the horizontal axis 576 as shown in FIG. 38. Specifically, therobotic arm 16 rotates the seed 12 with respect to the captured image550 until the longitudinal axis 552 is parallel to the horizontal axis576 (e.g., subject to a tolerance level such as within one degree ofparallelism). When the seed 12 is properly oriented, the procedure 1200continues to block 1222.

In block 1222, the controller 400 operates the camera 58 to captureanother image 580 of the gripped seed 12 from a side elevation (i.e.,the field of view of the camera 58), as shown in FIG. 39. The image 580is analyzed in block 1224 to identify the gripped seed 12 and a location596 of the hilum 424 of the seed 12 relative to a center of mass orlongitudinal axis of the seed 12. In the illustrative embodiment, thecontroller 400 utilizes blob detection to determine the location 598 ofthe seed 12 in the captured image 580. Additionally, the controller 400utilizes a suitable algorithm to determine the location of the hilum 424on the seed 12 in the capture image 580. For example, the controller 400may determine the location 596 of the hilum 424 using the referenceimage 536 of a hilum (see FIG. 32) and/or image feature matchingalgorithms as described above. The controller 400 identifies alongitudinal end 582 (e.g., either the left or right end) of the seed 12and a vertical slice 584 of the longitudinal end 582 of the seed 12 in amanner similar to that described above. As shown in FIG. 40, thecontroller 400 identifies a center of mass 586 of the vertical slice 584of the seed 12 and a center of mass 588 of the hilum 424 and draws avirtual line 590 between the centers of mass 586, 588. The controller400 further determines an angle 592 of the line 590 relative to ahorizontal axis 594 of the captured image 580 or the longitudinal axis552 of the seed 12.

Returning to FIG. 18, the procedure 1200 advances to block 1226 in whichthe controller 400 operates the robotic arm 16 to orient the seed 12 toalign the center of mass 588 of the hilum 424 with the longitudinal axis552 of the seed 12 as shown in FIG. 41. In particular, the robotic arm16 rotates the seed 12 toward or away from the camera 58 until the line590 between the centers of mass 586, 588 is parallel to the horizontalaxis 594 of the captured image 580. At that point, the line 590corresponds to the longitudinal axis 436 of the hilum 424 such that theseed plane 438 defined by the longitudinal axis 436 of the hilum 424 andthe longitudinal axis 436 of the seed 12 is aligned with a defined planeof the coordinate system of the robotic arm 16.

The procedure 1200 may then advance to block 1228 of FIG. 19. In block1228, the controller 400 operates the camera 58 to capture images of thegripped seed 12 from a side elevation. Such images 600 are shown inFIGS. 43-48. Returning to FIG. 19, in block 1230, the controller 400analyzes the captured image 600 to determine the location of theembryonic axis 440 of the seed 12. The controller 400 may use anysuitable algorithm for doing so.

For example, in the illustrative embodiment, the controller 400 mayutilize a reference image 612 of an embryonic axis, as shown in FIG. 42,in conjunction with the geometric object-identifying function and/orcorrelation model described above to identify the embryonic axis 440. Itwill be appreciated that the controller 400 has identified a match 602for the embryonic axis 440 in each of FIGS. 46-48 as shown. However, thecontroller 400 has failed to identify the embryonic axis 440 in each ofFIGS. 43-45, because a significant portion of the embryonic axis 440 isnot within the field of view of the camera 58. In those circumstances,the controller 400 operates the robotic arm 16 to rotate the seed 12until the embryonic axis 440 is within the field of view of the camera58 and detected by the controller 400.

Returning to FIG. 19, the procedure 1200 advances to block 1232 in whichthe controller 400 determines a location at which to trim the embryonicaxis 440 of the seed 12. To do so, the controller 400 determines thelocation 708 of the seed 12, the location 602 of the embryonic axis 440,and the location 710 of the hilum 424 of the seed 12 in the capturedimage 600 or a new image captured by the camera 58 as shown in FIGS.49-52. In particular, the controller 400 identifies an edge 620 of thehilum 424 nearest the embryonic axis 440 and an edge 622 of the seed 12on the same side as the embryonic axis 440 as shown in FIG. 51. Further,in the illustrative embodiment, the controller 400 determines a verticalcross section 624 halfway between the edges 620, 622. The vertical crosssection 624 corresponds with the location at which the system 10 is totrim the embryonic axis 440 of the seed 12. In other embodiments, thecontroller 400 may identify a point other than the midpoint between theedges 620, 622 (e.g., based on user input).

As indicated above, the controller 400 has calibrated the system 10 suchthat the coordinate system for the robotic arm 16 and the coordinatesystem of the camera 58 are mapped to one another. Because thecoordinate system of the robotic arm 16 is known, the controller 400knows the location of a center 626 of the grip 18 with respect to thecaptured image 600. The controller 400 also knows the correspondencebetween physical distance in the coordinate system of the robotic arm 16(e.g., in millimeters) and distance in the coordinate system of thecamera 58 (e.g., in pixels). That information is used to determine ahorizontal distance 628 between the center 626 and the vertical crosssection 624 in the captured image 600 as shown in FIG. 52. Thecontroller 400 further calculates a distance of the embryonic trimmingcut relative to the center 626 of the grip 18.

Returning to FIG. 19, in block 1234, the controller 400 determines theposition at which to place the gripped seed 12 on the cutting block 116of the cutting station 30 to be trimmed and bisected by the cuttingblade 170. To do so, the controller 400 operates the camera 56 tocapture an image 640 of the seed 12 from a bottom perspective. Asindicated above, the mapping between the coordinate systems of therobotic arm 16 and the camera 56 is known, so a point 642 projectedalong the grip axis 358 to the captured image 640 may be determined. Thecontroller 400 further analyzes the captured image 640 to identify aback edge 644 of the seed 12 (i.e., opposite the hilum 424 and theembryonic axis 440) and a distance 646 between the point 642 and theback edge 644 as shown in FIG. 53. Because the controller 400 has thelocation of the front wall 154 of the cutting block 116 stored inmemory, the controller 400 is able to properly position the seed 12 onthe cutting block 116. In particular, the controller 400 operates therobotic arm 16 to position the seed 12 on the flange 124 and with thecenter of the grip 18 defined by the grip axis 358 positioned thedetermined distance 646 away from the front wall 154.

Returning to FIG. 19, in block 1236, the controller 400 determines thedepth of the trimming and/or bisection cut and the positioning of theblade 170 for cutting the seed 12. As indicated above, the controller400 previously determined a point at which to trim the embryonic axis(i.e., the vertical cross section 624 as shown in FIG. 52). In theillustrative embodiment, the controller 400 maps the vertical crosssection 624 to a corresponding location 650 on the captured image 640,which was taken from a different perspective, by virtue of the knowncoordinate systems of each of the cameras 56, 58. Further, thecontroller 400 determines a width 652 of the seed 12 at thecorresponding location 650 as shown in FIG. 54. The controller 400 alsoidentifies the location of a back edge 644 of the seed 12. Based on thisinformation and the desired depth of the trim cut and/or bisection cut(e.g., from user inputs), the controller 400 is able to determine thedistance to move the cutting blade 170 toward the front wall 154 whentrimming and/or bisecting the embryonic axis 440.

The controller 400 also determines the appropriate positioning of thecutting blade 170 for the bisection of the seed 12. To do so, thecontroller 400 operates the camera 58 to capture an image 660 of theseed 12 and analyzes the captured image 660 to locate a center of mass662 of the embryonic axis 440 as shown in FIG. 55. As indicated above,the controller 400 may first determine the location 602 of the embryonicaxis 440 in the captured image 660 using, for example, a featurematching algorithm in conjunction with the reference image 612 (see FIG.42). Further, the controller 400 determines a distance 664 between abottom edge 666 of the seed 12 and the center of mass 662 of theembryonic axis 440. As indicated above, the controller 400 may convertthe pixel distance to a physical distance. Accordingly, the distance 664is used to determine the distance above the flange 124 at which thehorizontal bisection cut is made.

Referring back to FIG. 15, once the controller 400 determines the properorientations of the seed 12 for trimming and bisection, the procedure1000 advances to block 1020 of FIG. 16. In block 1020, the controller400 operates the robotic arm 16 to position the gripped seed 12 on thecutting block 116. As described above, based on structural data storedin memory, the controller 400 is able to determine the distance 646between the grip axis 358 and the back edge 644 of the gripped seed 12.Accordingly, in the illustrative embodiment, the controller 400 operatesthe robotic arm 16 to position the seed 12 on the flange 124 at a pointin which the grip axis 358 is positioned the determined distance 646away from the front wall 154 of the cutting block 116. At the distance646, the seed 12 is positioned for cutting at the proper depth andorientation. In the illustrative embodiment, the seed 12 is positionedsuch that the back edge 644 of the seed 12 just contacts the front wall154 of the cutting block 116.

In block 1022, the controller 400 operates the cutting device 112 totrim the embryonic axis 440. To do so, the controller 400 activates thecompressed air source 230 to cause the shaft 224 (and hence the jaws244, 246) to rotate about the axis 226. The shaft 224 is rotated toposition the cutting blade 170 vertically (i.e., perpendicular to theflange 124 of the cutting block 116). As shown in FIG. 56, the cuttingblade 170 is aligned with the slot 164 defined in the flange 124.

The controller 400 may also operate the intermediate drive stage 210 toraise or lower the cutting blade 170, as indicated by arrows 700 in FIG.56. To trim the embryonic axis 440, the controller 400 operates thedrive stage 194 of the cutting device 112 to advance the cutting blade170 linearly along the axis 226 toward the seed 12 on the block 116. Asshown in FIG. 57, the cutting blade 170 is advanced into the slot 164and the seed 12 until the cutting blade 170 reaches the previouslydetermined cutting distance (e.g., relative to the front wall 154),thereby trimming the embryonic axis 440.

As shown in FIG. 63, the cutting blade 170 is advanced through theembryonic axis 440 to separate the tip 442 of the axis 440 from the restof the axis 440. As described above, typically, between ⅓ and ½ of theembryonic axis 440 may left attached. In other words, between ½ and ⅔ ofthe embryonic axis 440 may be trimmed along with the tip 442 from therest of the embryonic axis 440. In the illustrative embodiment, thecutting blade 170 does not penetrate the cotyledons 412, 414 when theembryonic axis 440 is trimmed. In some embodiments, it may be desirableto wound the cotyledons 412, 414 by advancing the cutting blade 170further into the seed 10. The controller 400 may then operate the drivestage 194 to move the cutting blade 170 away from the seed 12 and out ofthe slot 164.

The procedure 1000 may then advance to block 1024 in which thecontroller 400 operates the cutting device 112 to position the cuttingblade 170 horizontally for bisection of the seed 12. To do so, thecontroller 400 activates the compressed air source 230 to cause theshaft 224 (and hence the jaws 244, 246) to rotate about the axis 226from the vertical position shown in FIGS. 56-57 to the horizontalposition shown in FIG. 58. The controller 400 may also operate theintermediate drive stage 210 to raise or lower the cutting blade 170 toalign the cutting blade 170 with the longitudinal axis 418 of the seed12. As discussed above, the controller 400 may use the distance 664 andother known physical dimensions to determine the distance above theflange 124 at which the cutting blade 170 is to be positioned.

In block 1026 of the procedure 1000, the controller 400 moves cuttingblade 170 toward the front wall 154 to bisect the seed 12. To do so, thecontroller 400 operates the drive stage 194 of the cutting device 112 toadvance the cutting blade 170 linearly along the axis 226 toward theseed 12 on the block 116. As shown in FIG. 59, the cutting blade 170 isadvanced into the seed 12 until the cutting blade 170 reaches thepreviously determined bisection distance as described above (e.g.,relative to the front wall 154).

As shown in FIG. 64, the cutting blade 170 is aligned with plane 438defined by the longitudinal axis 436 of the hilum 424 and thelongitudinal axis 418 of the seed 12 and advanced through the seed coat416 and the hilum 424 along the plane 438, thereby creating an opening702 in the seed 12. The embryonic axis 440 is sliced into a medialsection 704 attached to the cotyledon 412 and a lateral section 706attached to the cotyledon 414. As shown in FIG. 64, the cutting blade170 passes through the base 444 of the embryonic axis 440. It will beappreciated that, in the illustrative embodiment, the cutting blade 170does not completely bisect the seed 12 into two pieces. Rather, afterthe embryonic trimming and bisection, the seed 12 can still betransported by the grip 18 as a single piece. The controller 400 maythen operate the drive stage 194 to move the cutting blade 170 away fromthe seed 12.

In block 1028 of the procedure 1000, the controller 400 operates therobotic arm 16 to move the bisected seed 12 to the plate 38 located atthe corresponding receiving area 26. The controller 400 then deactivatesthe negative pressure source 356 to drop the bisected seed 12 onto theplate 38. In block 1030, the controller 400 operates the robotic arm 16to clear any debris from the cutting block 116. In some embodiments, therobotic arm 16 may perform one or more passes of the grip 18 along theupper wall 156 of the flange 124 to clear debris. In other embodiments,the grip assembly 320 includes a pressure source that is electricallycoupled to the controller 400 and configured to deliver pressurizedfluid (e.g., compressed air) through the passageways 352, 354 to repellight objects such as debris. In such embodiments, the controller 400may deliver operate the pressure source to deliver pressurized fluid tothe cutting block 116 as the grip 18 passes along the flange 124.

In block 1032, the controller 400 may operate the robotic arm 16 and thecutting device 112 to replace periodically the cutting blade 170.Depending on the particular embodiment, the cutting blade 170 may bereplaced after a predefined amount of time has lapsed, after a thresholdnumber of seeds 12 have been processed, and/or in response to anothercondition.

It will be appreciated that the procedure 1000 or portions of theprocedure 1000 may be repeated for each seed 12 on the plate 36 in thedelivery area 24. Further, the procedure 1000 may be implemented usingboth robotic arms 16 such that the arms 16 alternate use of the stations28, 30. Further, it should be appreciated that the procedure may beimplemented with one or more robotic arms 16 that each utilize its owndedicated stations 28, 30.

After one or more of the cut seeds 12 have been placed in a receivingarea 26, the user may remove the seeds 12 from the system 10 for furtherprocessing. Among other things, the user may remove separate thecotyledon from the seed coat, additionally wound the cotyledon, orinoculate the cotyledon with an Agrobacterium culture. To separate theseed coat 416 from the cotyledons 412, 414, the user may widen theopening 702 to further expose the cotyledons 412, 414. The cotyledons412, 414 may be removed from the seed coat 416, and the seed coat 416discarded. As shown in FIG. 65, each cotyledon, which may be referred toas a split soybean seed or cotyledon segment, includes a section of theembryonic axis. In the illustrative embodiment, the cotyledon segment412 includes the section 704 of the embryonic axis 440, while thecotyledon segment 414 includes the section 706 of the embryonic axis440. Each of the cotyledon segments 412, 414 is then ready for furtherprocessing, including additional wounding or inoculation with anAgrobacterium culture.

An Agrobacterium culture is a widely utilized method for introducing anexpression vector into plants is based on the natural transformationsystem of Agrobacterium. Horsch et al., Science 227:1229 (1985). A.tumefaciens and A. rhizogenes are plant pathogenic soil bacteria knownto be useful to genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of the plant. Kado, C. I., Crit.Rev. Plant. Sci. 10:1 (1991). Descriptions of Agrobacterium vectorsystems and methods for Agrobacterium-mediated gene transfer are alsoavailable, for example, Gruber et al., supra, Miki et al., supra,Moloney et al., Plant Cell Reports 8:238 (1989), and U.S. Pat. Nos.4,940,838 and 5,464,763.

If Agrobacterium is used for the transformation, the DNA to be insertedshould be cloned into special plasmids, namely either into anintermediate vector or into a binary vector. Intermediate vectors cannotreplicate themselves in Agrobacterium. The intermediate vector can betransferred into Agrobacterium tumefaciens by means of a helper plasmid(conjugation). The Japan Tobacco Superbinary system is an example ofsuch a system (reviewed by Komari et al. (2006) In: Methods in MolecularBiology (K. Wang, ed.) No. 343: Agrobacterium Protocols (2^(nd) Edition,Vol. 1) HUMANA PRESS Inc., Totowa, N.J., pp. 15-41; and Komori et al.(2007) Plant Physiol. 145:1155-1160). Binary vectors can replicatethemselves both in E. coli and in Agrobacterium. They comprise aselection marker gene and a linker or polylinker which are framed by theright and left T-DNA border regions. They can be transformed directlyinto Agrobacterium (Holsters, 1978). The Agrobacterium used as host cellis to comprise a plasmid carrying a vir region. The Ti or Ri plasmidalso comprises the vir region necessary for the transfer of the T-DNA.The vir region is necessary for the transfer of the T-DNA into the plantcell. Additional T-DNA may be contained.

The virulence functions of the Agrobacterium tumefaciens host willdirect the insertion of a T-strand containing the construct and adjacentmarker into the plant cell DNA when the cell is infected by the bacteriausing a binary T DNA vector (Bevan (1984) Nuc. Acid Res. 12:8711-8721)or the co-cultivation procedure (Horsch et al. (1985) Science227:1229-1231). Generally, the Agrobacterium transformation system isused to engineer dicotyledonous plants (Bevan et al. (1982) Ann. Rev.Genet 16:357-384; Rogers et al. (1986) Methods Enzymol. 118:627-641).The Agrobacterium transformation system may also be used to transform,as well as transfer, DNA to monocotyledonous plants and plant cells. SeeU.S. Pat. No. 5,591,616; Hernalsteen et al. (1984) EMBO J 3:3039-3041;Hooykass-Van Slogteren et al. (1984) Nature 311:763-764; Grimsley et al.(1987) Nature 325:1677-179; Boulton et al. (1989) Plant Mol. Biol.12:31-40; and Gould et al. (1991) Plant Physiol. 95:426-434.

Split soybean seeds comprising a portion of an embryonic axis may betypically inoculated with Agrobacterium culture containing a suitablegenetic construct for about 0.5 to 3.0 hours, more typically for about0.5 hours, followed by a period of co-cultivation on suitable medium forup to about 5 days. Explants that putatively contain a copy of thetransgene arise from the culturing of the transformed split soybeanseeds comprising a portion of an embryonic axis. These explants may beidentified and isolated for further tissue propagation.

A number of alternative techniques can also be used for inserting DNAinto a host plant cell. Those techniques include, but are not limitedto, transformation with T-DNA delivered by Agrobacterium tumefaciens orAgrobacterium rhizogenes as the transformation agent. From example ofAgrobacterium technology are described in, for example, in U.S. Pat. No.5,177,010, U.S. Pat. No. 5,104,310, European Patent Application No.0131624B1, European Patent Application No. 120516, European PatentApplication No. 159418B1 , European Patent Application No. 176112, U.S.Pat. No. 5,149,645, U.S. Pat. No. 5,469,976, U.S. Pat. No. 5,464,763,U.S. Pat. No. 4,940,838, U.S. Pat. No. 4,693,976, European PatentApplication No. 116718, European Patent Application No. 290799, EuropeanPatent Application No. 320500, European Patent Application No. 604662,European Patent Application No. 627752, European Patent Application No.0267159, European Patent Application No. 0292435, U.S. Pat. No.5,231,019, U.S. Pat. No. 5,463,174, U.S. Pat. No. 4,762,785, U.S. Pat.No. 5,004,863, and U.S. Pat. No. 5,159,135. The use of T-DNA-containingvectors for the transformation of plant cells has been intensivelyresearched and sufficiently described in European Patent Application120516; An et al, (1985, EMBO J. 4:277-284), Fraley et al, (1986, Crit.Rev. Plant Sci. 4: 1-46), and Lee and Gelvin (2008, Plant Physiol. 146:325- 332), and is well established in the field.

Another known method of plant transformation is microprojectile-mediatedtransformation wherein DNA is carried on the surface ofmicroprojectiles. In this method, the expression vector is introducedinto plant tissues with a biolistic device that accelerates themicroprojectiles to speeds sufficient to penetrate plant cell walls andmembranes. Sanford et al., Part. Sci. Technol. 5:27 (1987), Sanford, J.C., Trends Biotech. 6:299 (1988), Sanford, J. C., Physiol. Plant 79:206(1990), Klein et al., Biotechnology 10:268 (1992).

Alternatively, gene transfer and transformation methods include, but arenot limited to, protoplast transformation through calcium chlorideprecipitation, polyethylene glycol (PEG)- or electroporation-mediateduptake of naked DNA (see Paszkowski et al. (1984) EMBO J 3:2717-2722,Potrykus et al. (1985) Molec. Gen. Genet. 199:169-177; Fromm et al.(1985) Proc. Nat. Acad. Sci. USA 82:5824-5828; and Shimamoto (1989)Nature 338:274-276) and electroporation of plant tissues (D'Halluin etal. (1992) Plant Cell 4:1495-1505).

While the disclosure has been illustrated and described in detail in thedrawings and foregoing description, such an illustration and descriptionis to be considered as exemplary and not restrictive in character, itbeing understood that only illustrative embodiments have been shown anddescribed and that all changes and modifications that come within thespirit of the disclosure are desired to be protected.

There are a plurality of advantages of the present disclosure arisingfrom the various features of the method, apparatus, and system describedherein. It will be noted that alternative embodiments of the method,apparatus, and system of the present disclosure may not include all ofthe features described yet still benefit from at least some of theadvantages of such features. Those of ordinary skill in the art mayreadily devise their own implementations of the method, apparatus, andsystem that incorporate one or more of the features of the presentinvention and fall within the spirit and scope of the present disclosureas defined by the appended claims.

1. A method for imaging a seed, the method comprising: using a roboticarm to position a seed for imaging, capturing a plurality of images ofthe seed, identifying a location of the embryo of the seed based on theplurality of captured images, identifying an edge of the hilum nearestthe identified location based on the plurality of captured images,identifying a point between the edge of the hilum and the outer edge ofthe seed at which to trim an embryonic axis of the seed, and moving theseed with the robotic arm to orient the seed in a position basedidentified point at which to trim the embryonic axis of the seed.
 2. Themethod of claim 1, wherein identifying the location of the embryo of theseed includes analyzing the captured images to locate the embryonic axisof the seed.
 3. The method of claim 2, wherein identifying the pointbetween the edge of the hilum and the outer edge of the seed at which totrim the embryonic axis of the seed includes identifying the edge of thehilum nearest the embryonic axis.
 4. The method of claim 3, whereinidentifying the point between the edge of the hilum and the outer edgeof the seed at which to trim the embryonic axis of the seed includesidentifying the outer edge of the seed on the same side of the seed asthe embryonic axis.
 5. The method of claim 1, wherein using the roboticarm to position the seed for imaging includes engaging the seed with agrip of the robotic arm.
 6. The method of claim 5, further comprisingcalculating a distance between the identified point at which to trim theembryonic axis of the seed and a center point of the grip.
 7. The methodof claim 5, wherein engaging the seed with the grip of the robotic armincludes attaching the seed to the grip via a suction force.
 8. A methodfor imaging a seed, the method comprising: using a robotic arm toposition a hydrated seed for imaging, capturing a plurality of images ofthe hydrated seed, determining an orientation of the hydrated seed and alocation of the hilum of the hydrated seed based on the plurality ofcaptured images, and moving the hydrated seed with a robotic arm toorient the hydrated seed in a position for cutting the seed based on thedetermined orientation of the hydrated seed and the location of thehilum.
 9. The method of claim 8, further comprising analyzing thecaptured images to locate an embryonic axis of the seed.
 10. The methodof claim 9, wherein determining the orientation of the hydrated seed andthe location of the hilum of the hydrated seed includes identifying anedge of the hilum nearest the embryonic axis.
 11. The method of claim10, wherein determining the orientation of the hydrated seed and thelocation of the hilum of the hydrated seed includes identifying theouter edge of the seed on the same side of the seed as the embryonicaxis.
 12. The method of claim 9, wherein using the robotic arm toposition the hydrated seed for imaging includes engaging the seed with agrip of the robotic arm.
 13. The method of claim 12, wherein engagingthe seed with the grip of the robotic arm includes attaching the seed tothe grip via a suction force.